Therapeutic or prophylactic agent for abnormal angiogenesis, and its applications

Abstract

To provide a novel therapeutic or prophylactic agent for abnormal angiogenesis, and its applications.SOLUTION: A therapeutic or prophylactic agent for abnormal angiogenesis is used that contains a lysine demethylase 3A inhibitor as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a therapeutic or preventive agent for abnormal angiogenesis and use thereof. [Background technology]

[0002] Angiogenesis can occur in vivo under both physiological and pathological conditions. Under physiological conditions, for example, normal angiogenesis occurs in fetal tissues (e.g., retina), which results in the formation of normal tissues. On the other hand, under pathological conditions, for example, abnormal angiogenesis occurs in disease-affected areas (e.g., retinal ischemic disease, cancer, retinopathy of prematurity, age-related macular degeneration, diabetic macular edema, pathological myopia, macular edema associated with retinal vein occlusion, and neovascular glaucoma), which results in the onset or aggravation of the disease.

[0003] Examples of abnormal angiogenesis include the formation of ischemic areas (in other words, the formation of areas where blood vessels are not formed) and the formation of abnormal blood vessels (in other words, the formation of blood vessels that run in an uncontrolled manner).

[0004] The formation of ischemic area and abnormal blood vessels may occur alone or in combination. For example, in age-related macular degeneration and pathological myopia, abnormal blood vessels are formed alone. On the other hand, in retinal ischemic disease, cancer, retinopathy of prematurity, diabetic macular edema, macular edema associated with retinal vein occlusion, and neovascular glaucoma, ischemic area is formed first, and then abnormal blood vessels are formed.

[0005] Anti-VEGF drugs are currently used as therapeutic agents for abnormal angiogenesis (see, for example, Non-Patent Documents 1 and 2). Non-Patent Documents 1 and 2 describe the use of aflibercept, bevacizumab, ranibizumab, and faricimab for the treatment of diabetic macular edema. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] N Eng J Med., 2015 March 26; 372(13): 1193-1203, "Aflibercept, Bevacizumab, or Ranibizumab for Diabetic Macular Edema" [Non-Patent Document 2] Charles C Wykoff et al., Lancet., 2022 Feb 19; 399(10326): 741-755, "Efficacy, durability, and safety of intravitreal faricimab with extended dosing up to every 16 weeks in patients with diabetic macular oedema(YOSEMITE and RHINE): two randomized, double-masked, phase 3 trials" Summary of the Invention [Problem to be solved by the invention]

[0007] However, the above-mentioned conventional techniques have a problem in that the therapeutic effect on abnormal angiogenesis is insufficient.

[0008] For example, the above-mentioned conventional technologies have room for improvement in that: (i) they only have the effect of temporarily reducing abnormal angiogenesis, which raises concerns about the need for frequent administration; (ii) they affect both normal and abnormal angiogenesis, which raises concerns about side effects; and (iii) anti-VEGF drugs inhibit angiogenesis, which raises concerns about the inability to achieve a sufficient therapeutic effect against the formation of ischemic areas.

[0009] An object of one aspect of the present invention is to provide a novel agent for treating or preventing abnormal angiogenesis, and use thereof. [Means for solving the problem]

[0010] One aspect of the present invention is the following: [1] A therapeutic or preventive agent for abnormal angiogenesis, comprising a lysine demethylase 3A inhibitor as an active ingredient.

[0011] [2] The agent for treating or preventing abnormal angiogenesis according to [1], wherein the lysine demethylase 3A inhibitor is a compound represented by the following chemical formula 1, a derivative thereof, or a salt thereof;

[0012] [ka]

[0013] [In Chemical Formula 1, R is hydrogen or any organic group].

[0014] [3] The agent for treating or preventing abnormal angiogenesis according to [1], wherein R is hydrogen, an alkyl group, an aryl group, a heterocyclic group, a fluoroalkyl group, an alkenyl group, a fluoroalkenyl group, an alkynyl group, a fluoroalkynyl group, an alkoxyl group, a fluoroalkoxyl group, an acetyl group, a carboxyalkyl group, or an alkylamide group.

[0015] [4] The therapeutic or preventive agent for abnormal angiogenesis according to [1], wherein the abnormal angiogenesis is the formation of an ischemic area and / or the formation of abnormal blood vessels.

[0016] [5] The therapeutic or preventive agent for abnormal angiogenesis according to any one of [1] to [4], wherein the abnormal angiogenesis is abnormal angiogenesis associated with retinal ischemic disease, cancer, retinopathy of prematurity, age-related macular degeneration, diabetic macular edema, pathological myopia, macular edema associated with retinal vein occlusion, or neovascular glaucoma.

[0017] [6] Use of a lysine demethylase 3A inhibitor for the manufacture of an agent for treating or preventing abnormal angiogenesis.

[0018] [7] A compound represented by the following chemical formula 1, a derivative thereof, or a salt thereof;

[0019] [ka]

[0020] [In Chemical Formula 1, R is hydrogen or any organic group].

[0021] [8] The compound, derivative, or salt according to [7], wherein R is a hydrogen atom, an alkyl group, an aryl group, a heterocyclic group, a fluoroalkyl group, an alkenyl group, a fluoroalkenyl group, an alkynyl group, a fluoroalkynyl group, an alkoxyl group, a fluoroalkoxyl group, an acetyl group, a carboxyalkyl group, or an alkylamide group.

[0022] [9] A contacting step of contacting a lysine demethylase 3A protein with a candidate substance for a therapeutic or preventive agent for abnormal angiogenesis; a measuring step of measuring the activity of the lysine demethylase 3A protein after the contacting step; A screening method for a therapeutic or preventive agent for abnormal angiogenesis, comprising a determination step of determining whether the candidate substance is a therapeutic or preventive agent for abnormal angiogenesis by comparing the activity of the lysine demethylase 3A protein measured in the measurement step with a control activity.

[0023]

[10] The measuring step comprises: a conversion step of contacting the lysine demethylase 3A protein with an excitable or luminescent donor bead to which a substrate peptide of the lysine demethylase 3A protein is bound, thereby converting the substrate peptide into a reaction product; an approaching step of bringing the donor bead to which the reaction product is bound into contact with a luminescent or excitable acceptor bead to which a substance capable of binding to the reaction product is bound, thereby bringing the donor bead and the acceptor bead into close proximity; The method for screening a therapeutic or preventive agent for abnormal angiogenesis described in

[10] , comprising a detection step of detecting light emitted from the donor bead or the acceptor bead upon approaching each other, wherein one of the donor bead and the acceptor bead is excitable and the other is luminescent, and detecting light emitted from the donor bead or the acceptor bead upon approaching each other.

[0024]

[11] The method for screening a therapeutic or preventive agent for abnormal angiogenesis according to [9], further comprising, after the determination step, an administration step of administering the candidate substance to an abnormal angiogenesis model animal.

[0025]

[12] The method for screening a therapeutic or preventive agent for abnormal angiogenesis according to

[11] , wherein the abnormal angiogenesis model animal is an OIR model mouse, an RVO (Retinal Vein Occlusion) model mouse, or an RVO model rabbit. Effect of the Invention

[0026] According to one aspect of the present invention, it is possible to provide a new agent for treating or preventing abnormal angiogenesis, and use thereof. [Brief description of the drawings]

[0027] [Figure 1] Graph 101 shows the inhibitory activity of TAB047 according to an embodiment of the present invention against lysine demethylase 3A, and graph 102 shows the inhibitory activity of TAB050 according to an embodiment of the present invention against lysine demethylase 3A. [Diagram 2] 201 is an image showing the therapeutic and preventive effects of TAB047 according to an embodiment of the present invention on abnormal angiogenesis, and 202 is a graph showing the therapeutic and preventive effects of TAB047 according to an embodiment of the present invention on abnormal angiogenesis. [Diagram 3] 301 is an image showing the therapeutic and preventive effects of TAB050 according to an embodiment of the present invention on abnormal angiogenesis, and 302 and 303 are graphs showing the therapeutic and preventive effects of TAB050 according to an embodiment of the present invention on abnormal angiogenesis. [Figure 4] 401 is a graph showing the results of a metabolic stability test of TAB047 according to an embodiment of the present invention, and 402 is a graph showing the results of a metabolic stability test of the hydrochloride salt of TAB047 according to an embodiment of the present invention. [Diagram 5] 501 is a graph showing the results of a metabolic stability test of TAB050 according to an embodiment of the present invention, and 502 is a graph showing the results of a metabolic stability test of the hydrochloride salt of TAB050 according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to each configuration described below, and various modifications are possible within the scope of the claims, and embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. In addition, all academic literature and patent documents described in this specification are incorporated herein by reference. In addition, unless otherwise specified in this specification, "A to B" representing a numerical range means "A or more, B or less."

[0029] The present invention can also contribute to achieving Goal 3 of the Sustainable Development Goals (SDGs) advocated by the United Nations, which is to "Ensure good health and promote well-being for all."

[0030] 1. Technical Concept of the Present Invention In vivo, normal angiogenesis occurs under physiological conditions, whereas abnormal angiogenesis occurs under pathological conditions. To form a normal organism, it is necessary to suppress abnormal angiogenesis while allowing normal angiogenesis to occur.

[0031] The present inventors have conducted research based on the original hypothesis that a substance capable of returning the pathological conditions that cause abnormal angiogenesis to the physiological conditions that cause normal angiogenesis would be useful as a new therapeutic or preventive agent for abnormal angiogenesis.

[0032] Specifically, the present inventors screened for new candidate substances for treating and preventing abnormal angiogenesis based on the unique hypothesis that substances that target proteins that control the expression of genes subject to epigenetic expression control are substances that can return pathological conditions that cause abnormal angiogenesis to physiological conditions that cause normal angiogenesis.

[0033] Many proteins are known to regulate the expression of genes that are subject to epigenetic expression control. The present inventors have found that among these proteins, a substance that inhibits the activity of lysine demethylase 3A is particularly useful as a new agent for treating and preventing abnormal angiogenesis, and have thus completed the present invention.

[0034] 2. Therapeutic or preventive agents for abnormal angiogenesis A therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention contains a lysine demethylase 3A inhibitor as an active ingredient.

[0035] The therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention has the following advantages: (i) since it has the effect of fundamentally treating or preventing abnormal angiogenesis, there is no need to worry about frequent administration; (ii) since it does not affect normal angiogenesis, there is no need to worry about side effects; and (iii) since it does not suppress angiogenesis like anti-VEGF drugs, it has a sufficient therapeutic effect on the formation of ischemic areas.

[0036] Lysine demethylase 3A is an enzyme that demethylates the methyl group of a specific lysine residue contained in histone. This demethylation partially loosens the chromatin structure formed by DNA and histone, making it easier for genes near the site where the histone is demethylated to be expressed. Lysine demethylase 3A inhibitors inhibit the above-mentioned process, thereby suppressing the expression of specific genes. Therefore, it is believed that the therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention acts on the expression of genes that are subject to epigenetic expression control.

[0037] As used herein, the term "therapeutic agent" refers to a drug that provides a therapeutic effect. The above therapeutic effect refers to, but is not limited to, the effects exemplified below.

[0038] (1) The severity of abnormal angiogenesis is reduced compared to when the therapeutic agent is not administered.

[0039] (2) preventing an increase in severity or progression of abnormal angiogenesis compared to when the therapeutic agent is not administered;

[0040] (3) reducing the rate at which the severity of abnormal angiogenesis increases or progresses, compared to when the therapeutic agent is not administered;

[0041] As used herein, the term "prophylactic agent" refers to a drug that provides a prophylactic effect. The prophylactic effect includes, but is not limited to, the effects exemplified below.

[0042] (1) Preventing the occurrence of abnormal angiogenesis or reducing the risk compared to when the prophylactic agent is not administered.

[0043] (2) Preventing or reducing the risk of recurrence of abnormal angiogenesis compared to when the prophylactic agent is not administered.

[0044] (3) preventing or reducing the risk of symptoms of abnormal angiogenesis compared to when the prophylactic agent is not administered;

[0045] The abnormal angiogenesis may be systemic or local.

[0046] The lysine demethylase 3A inhibitor can be a nucleic acid (eg, DNA, RNA, or antisense RNA), a protein (eg, an antibody), a polymeric compound, or a small molecule compound.

[0047] The lysine demethylase 3A inhibitor is preferably a compound represented by the following chemical formula 1, a derivative thereof, or a salt thereof. With this composition, abnormal angiogenesis can be effectively treated or prevented.

[0048] [ka]

[0049] [In Chemical Formula 1, R is hydrogen or any organic group].

[0050] The R is preferably hydrogen, an alkyl group, an aryl group, a heterocyclic group, a fluoroalkyl group, an alkenyl group, a fluoroalkenyl group, an alkynyl group, a fluoroalkynyl group, an alkoxyl group, a fluoroalkoxyl group, an acetyl group, a carboxyalkyl group, or an alkylamide group. With this configuration, abnormal angiogenesis can be effectively treated or prevented.

[0051] When R is other than hydrogen, R can have, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 carbon atoms.

[0052] When R contains an atom other than carbon, the atom may be, for example, N, O, S, B, Si, P, F, Cl, Br, and / or I. The number of these atoms contained in R is not limited, and may be, for example, a total of 1 to 10, a total of 1 to 5, or a total of 1 to 3.

[0053] When R is an alkyl group, the alkyl group may be a straight-chain alkyl group or a branched alkyl group. For example, the alkyl group may be a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, a sec-butyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a neopentyl group, a sec-pentyl group, a 3-pentyl group, a tert-pentyl group, or a cyclopentyl group.

[0054] As will be apparent from the examples described later, the therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention can adjust the membrane permeability (in other words, bioabsorbability) and metabolic stability to desired properties by appropriately selecting R.

[0055] A therapeutic or prophylactic agent having high membrane permeability (in other words, bioabsorbability) has the advantage of having a high therapeutic effect, whereas a therapeutic or prophylactic agent having high metabolic stability has the advantage of having a sustained pharmacological effect.

[0056] For example, it is possible to enhance the membrane permeability (in other words, bioabsorbability) of the therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention by selecting a lipophilic functional group as R. Examples of such R include functional groups other than hydrogen (e.g., methyl group, ethyl group, propyl group, isopropyl group, cyclopropyl group, butyl group, sec-butyl group, tert-butyl group, cyclobutyl group, pentyl group, neopentyl group, sec-pentyl group, 3-pentyl group, tert-pentyl group, and cyclopentyl group).

[0057] The above-mentioned derivatives and salts may have any structure as long as they have an inhibitory activity against lysine demethylase 3A. Whether or not a specific substance has an inhibitory activity against lysine demethylase 3A can be determined according to a known method. For example, after mixing a specific substance with lysine demethylase 3A, it is examined whether or not the methyl group of a specific lysine residue contained in histone is demethylated by the lysine demethylase 3A. If the methyl group of a specific lysine residue contained in histone is not demethylated by the lysine demethylase 3A in the test, it can be determined that the specific substance has an inhibitory activity against lysine demethylase 3A.

[0058] As used herein, the term "derivative" refers to a group of compounds that are produced by replacing a portion of a molecule of a specific compound with another functional group or another atom.

[0059] Examples of the other functional groups include an alkyl group, an alkoxy group, an alkylthio group, an aryl group, an aryloxy group, an arylthio group, an arylalkyl group, an arylalkoxy group, an arylalkylthio group, an arylalkenyl group, an arylalkynyl group, an allyl group, an amino group, a substituted amino group, a silyl group, a substituted silyl group, a silyloxy group, a substituted silyloxy group, an arylsulfonyloxy group, an alkylsulfonyloxy group, a nitro group, etc. Examples of the other atoms include a carbon atom, a hydrogen atom, an oxygen atom, a nitrogen atom, a sulfur atom, a phosphorus atom, a halogen atom, etc.

[0060] In this specification, the term "salt" refers to any physiologically acceptable salt that can be administered to a subject as a pharmaceutical, and is not limited thereto. Examples of the salt include alkali metal salts (potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), ammonium salts, organic base salts (trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.), organic acid salts (acetate salt, maleate salt, tartrate salt, methanesulfonate salt, benzenesulfonate salt, formate salt, toluenesulfonate salt, trifluoroacetate salt, etc.), inorganic acid salts (hydrochloride salt, hydrobromide salt, sulfate salt, phosphate salt, etc.), etc.

[0061] The type of abnormal angiogenesis is not limited. The abnormal angiogenesis may be the formation of an ischemic region (in other words, the formation of a region where blood vessels are not formed) and / or the formation of abnormal blood vessels (in other words, the formation of blood vessels running in a disorderly manner). The abnormal angiogenesis may be abnormal angiogenesis including the formation of an ischemic region, abnormal angiogenesis including the formation of abnormal blood vessels, or abnormal angiogenesis including both the formation of an ischemic region and the formation of abnormal blood vessels.

[0062] The therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention uses a mechanism of action completely different from that of conventional anti-VEGF drugs and does not suppress angiogenesis. Therefore, as shown in the examples described below, the therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention can treat and prevent not only the formation of abnormal blood vessels but also the formation of ischemic regions.

[0063] The disease in which the abnormal angiogenesis occurs is not limited. The abnormal angiogenesis may be associated with retinal ischemic disease, cancer, retinopathy of prematurity, age-related macular degeneration, diabetic macular edema, pathological myopia, macular edema associated with retinal vein occlusion, or neovascular glaucoma. The abnormal angiogenesis may be associated with retinal ischemic disease, cancer, retinopathy of prematurity, diabetic macular edema, macular edema associated with retinal vein occlusion, or neovascular glaucoma, which are accompanied by the formation of ischemic areas. The abnormal angiogenesis may be associated with age-related macular degeneration or pathological myopia, which are accompanied by the formation of abnormal blood vessels and are not accompanied by the formation of ischemic areas.

[0064] The therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention can treat or prevent abnormal angiogenesis associated with the above-mentioned diseases. Furthermore, the therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention can treat or prevent increased vascular permeability and ischemia caused by the above-mentioned diseases.

[0065] The abnormal angiogenesis treatment or prevention agent according to one embodiment of the present invention can be administered to a subject by any administration route. Examples of the administration route include parenteral administration (e.g., ophthalmic administration, intraconjunctival administration, intravitreal administration, subconjunctival administration, subtenon administration, intraperitoneal administration), oral administration, transdermal administration, transmucosal administration, and intravenous administration. Therefore, the dosage form of the abnormal angiogenesis treatment or prevention agent according to one embodiment of the present invention can be eye drops, oral medication, topical medication, injection, inhalant, eye drops, etc.

[0066] The amount of an active ingredient contained in the therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention is not particularly limited, and may be, for example, 0.00001% by mass to 100% by mass, 0.0001% by mass to 100% by mass, 0.001% by mass to 100% by mass, 0.01% by mass to 100% by mass, 0.1% by mass to 100% by mass, or 0.1% by mass to 100% by mass. The content may be 0.1% to 95% by mass, 0.1% to 90% by mass, 0.1% to 80% by mass, 0.1% to 70% by mass, 0.1% to 60% by mass, 0.1% to 50% by mass, 0.1% to 40% by mass, 0.1% to 30% by mass, 0.1% to 20% by mass, or 0.1% to 10% by mass.

[0067] When the agent for treating or preventing abnormal angiogenesis according to one embodiment of the present invention is in a liquid form, the amount of the active ingredient contained in the agent for treating or preventing abnormal angiogenesis according to one embodiment of the present invention is not particularly limited, and may be, for example, 1 μM to 100 mM, 10 μM to 100 mM, 100 μM to 100 mM, 1 mM to 100 mM, or 1 mM to 10 mM.

[0068] The agent for treating or preventing abnormal angiogenesis according to one embodiment of the present invention may contain ingredients other than the above-mentioned active ingredients.

[0069] The components other than the above-mentioned active ingredient are not particularly limited, and may be, for example, a buffering agent, a pH adjusting agent, an isotonicity agent, a preservative, an antioxidant, a high molecular weight polymer, an excipient, a solvent, an antibacterial agent, and the like.

[0070] Examples of the buffer include phosphoric acid or a phosphate salt, boric acid or a borate salt, citric acid or a citrate salt, acetic acid or an acetate salt, carbonic acid or a carbonate salt, tartaric acid or a tartrate salt, ε-aminocaproic acid, and trometamol. Examples of the phosphate salt include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. Examples of the borate salt include borax, sodium borate, and potassium borate. Examples of the citrate salt include sodium citrate, disodium citrate, and trisodium citrate. Examples of the acetate salt include sodium acetate and potassium acetate. Examples of the carbonate salt include sodium carbonate and sodium bicarbonate. Examples of the tartrate salt include sodium tartrate and potassium tartrate.

[0071] Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0072] Examples of the isotonicity agent include ionic isotonicity agents (eg, sodium chloride, potassium chloride, calcium chloride, magnesium chloride) and non-ionic isotonicity agents (eg, glycerin, propylene glycol, sorbitol, mannitol).

[0073] Examples of the preservative include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0074] Examples of the antioxidant include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0075] Examples of the high molecular weight polymer include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

[0076] Examples of the excipient include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

[0077] Examples of the solvent include water, physiological saline, and alcohol.

[0078] Examples of the antibacterial agents include β-lactam, aminoglycoside, tetracycline, lincomycin, chloramphenicol, macrolide, ketolide, polypeptide, and glycopeptide antibiotics; and pyridonecarboxylic acid (quinolone), new quinolone, oxazolidinone, and sulfa drug-based synthetic antibacterial agents.

[0079] The amount of ingredients other than the active ingredient contained in the therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention is not particularly limited, and may be, for example, 0% by mass to 99.99999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.999% by mass, 0% by mass to 99.99% by mass, 5% by mass to 99. It may be 9% by mass, 10% by mass to 99.9% by mass, 20% by mass to 99.9% by mass, 30% by mass to 99.9% by mass, 40% by mass to 99.9% by mass, 50% by mass to 99.9% by mass, 60% by mass to 99.9% by mass, 70% by mass to 99.9% by mass, 80% by mass to 99.9% by mass, or 90% by mass to 99.9% by mass.

[0080] The administration interval of the therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention is not limited, and may be, for example, once every 12 hours, once a day, once every two days, once every three days, once every four days, or once every five days. The administration period of the therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention is not limited, and may be, for example, 3 days, 5 days, 7 days, 10 days, 15 days, or 30 days. The therapeutic or preventive agent according to one embodiment of the present invention can fundamentally treat or prevent abnormal angiogenesis, and therefore does not require excessively frequent administration.

[0081] The subjects of administration of the therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention are not limited, and examples thereof include humans and non-human animals (e.g., livestock, pets, and laboratory animals). Examples of non-human animals include monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.

[0082] 3. Use of Lysine Demethylase 3A Inhibitors The matters explained in the above section "2. Agent for treating or preventing abnormal angiogenesis" will not be explained further below.

[0083] One embodiment of the present invention relates to the use of a lysine demethylase 3A inhibitor for the manufacture of an agent for the treatment or prevention of abnormal angiogenesis.

[0084] The lysine demethylase 3A inhibitor is preferably a compound represented by the following chemical formula 1, a derivative thereof, or a salt thereof.

[0085] [ka]

[0086] [In Chemical Formula 1, R is hydrogen or any organic group].

[0087] The above R is preferably a hydrogen atom, an alkyl group, an aryl group, a heterocyclic group, a fluoroalkyl group, an alkenyl group, a fluoroalkenyl group, an alkynyl group, a fluoroalkynyl group, an alkoxyl group, a fluoroalkoxyl group, an acetyl group, a carboxyalkyl group, or an alkylamide group.

[0088] The type of abnormal angiogenesis is not limited. The abnormal angiogenesis may be the formation of an ischemic region (in other words, the formation of a region where blood vessels are not formed) and / or the formation of abnormal blood vessels (in other words, the formation of blood vessels running in a disorderly manner). The abnormal angiogenesis may be abnormal angiogenesis including the formation of an ischemic region, abnormal angiogenesis including the formation of abnormal blood vessels, or abnormal angiogenesis including both the formation of an ischemic region and the formation of abnormal blood vessels.

[0089] The disease in which the abnormal angiogenesis occurs is not limited. The abnormal angiogenesis may be associated with retinal ischemic disease, cancer, retinopathy of prematurity, age-related macular degeneration, diabetic macular edema, pathological myopia, macular edema associated with retinal vein occlusion, or neovascular glaucoma. The abnormal angiogenesis may be associated with retinal ischemic disease, cancer, retinopathy of prematurity, diabetic macular edema, macular edema associated with retinal vein occlusion, or neovascular glaucoma, which are accompanied by the formation of ischemic areas. The abnormal angiogenesis may be associated with age-related macular degeneration or pathological myopia, which are accompanied by the formation of abnormal blood vessels and are not accompanied by the formation of ischemic areas.

[0090] The method for producing the agent for treating or preventing abnormal angiogenesis is not limited, and any method generally used in the art may be used. For example, the agent for treating or preventing abnormal angiogenesis may be produced by mixing the above-mentioned active ingredient with, if necessary, an ingredient other than the above-mentioned active ingredient.

[0091] [4. Compounds, their derivatives, or their salts] The matters explained in the above section "2. Agent for treating or preventing abnormal angiogenesis" will not be explained further below.

[0092] A compound, a derivative thereof, or a salt thereof according to one embodiment of the present invention is a compound, a derivative thereof, or a salt thereof represented by the following chemical formula 1:

[0093] [ka]

[0094] [In Chemical Formula 1, R is hydrogen or any organic group].

[0095] The compound according to one embodiment of the present invention, its derivative, or its salt can be used as an active ingredient of a therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention.

[0096] The above R is preferably a hydrogen atom, an alkyl group, an aryl group, a heterocyclic group, a fluoroalkyl group, an alkenyl group, a fluoroalkenyl group, an alkynyl group, a fluoroalkynyl group, an alkoxyl group, a fluoroalkoxyl group, an acetyl group, a carboxyalkyl group, or an alkylamide group.

[0097] The above R, derivatives, salts, etc. have been explained in the above section [2. Agent for treating or preventing abnormal angiogenesis], so the explanation thereof will be omitted here.

[0098] The compound according to one embodiment of the present invention, its derivative, or its salt can be synthesized according to a known synthesis method, an example of which is shown below.

[0099] [ka]

[0100] (i) According to the above synthesis scheme, a compound according to an embodiment of the present invention in which R is a methyl group can be synthesized. (ii) By demethylating a compound according to an embodiment of the present invention in which R is a methyl group, a compound according to an embodiment of the present invention in which R is hydrogen can be synthesized. (iii) In the above synthesis scheme, by subjecting compound A to reaction 2 instead of compound B, a compound according to an embodiment of the present invention in which R is hydrogen can be synthesized. (iv) In reaction 1, by replacing "MeOH" with another substance (e.g., another alcohol), a compound according to an embodiment of the present invention having various R can be synthesized. (v) By subjecting a commercially available compound in which "OMe" of compound B is substituted to reaction 2, a compound according to an embodiment of the present invention having various R can be synthesized. As shown in the examples described later, a compound according to an embodiment of the present invention can be synthesized by click chemistry.

[0101] 5. Screening method for therapeutic or preventive agents for abnormal angiogenesis A screening method for a therapeutic or preventive agent for abnormal angiogenesis according to one embodiment of the present invention comprises a contacting step of contacting a lysine demethylase 3A protein with a candidate substance for a therapeutic or preventive agent for abnormal angiogenesis, a measuring step of measuring the activity of the lysine demethylase 3A protein after the contacting step, and a determination step of determining whether the candidate substance is a therapeutic or preventive agent for abnormal angiogenesis by comparing the activity of the lysine demethylase 3A protein measured in the measuring step with a control activity.

[0102] The candidate substance for the treatment or prevention of abnormal angiogenesis is not limited, and may be, for example, an organic compound or an inorganic compound. The candidate substance for the treatment or prevention of abnormal angiogenesis may be, for example, a nucleic acid (e.g., DNA, RNA, or antisense RNA), a protein (e.g., an antibody), a polymer compound, or a low molecular weight compound.

[0103] In the contacting step, the lysine demethylase 3A protein is contacted with a candidate substance for a therapeutic or preventive agent for abnormal angiogenesis.

[0104] The specific configuration of the contact step is not limited. In the contact step, for example, (A) the lysine demethylase 3A protein and the candidate substance may be added to a desired reaction solution in a test tube, and the lysine demethylase 3A protein and the candidate substance may be contacted in the reaction solution, or (B) the candidate substance may be added to a culture solution in which cells expressing the lysine demethylase 3A protein are cultured, and the lysine demethylase 3A protein and the candidate substance may be contacted in the cells.

[0105] The contacting step is followed by a measuring step of measuring the activity of the lysine demethylase 3A protein.

[0106] The specific configuration of the measurement step is not limited. For example, the measurement step may be (C) after the above-mentioned step (A), a substrate of the lysine demethylase 3A protein (e.g., a methylated polypeptide or a methylated histone) is added to the reaction solution to measure whether the substrate is demethylated, or (D) after the above-mentioned step (B), to measure whether the methylated histone in the cell is demethylated.

[0107] In the measuring step, the activity of the lysine demethylase 3A protein may be measured by AlphaScreen® Assay. According to this configuration, the activity of the lysine demethylase 3A protein can be measured with high sensitivity and ease, and it is also possible to determine with high sensitivity and ease whether the candidate substance is a therapeutic or preventive agent for abnormal angiogenesis.

[0108] The AlphaScreen Assay can be performed using a commercially available kit (eg, AlphaLISA (registered trademark) and AlphaScreen (registered trademark) manufactured by PerkinElmer).

[0109] The measurement step may include a conversion step of contacting the lysine demethylase 3A protein with an excitable or luminescent donor bead to which a substrate peptide of the lysine demethylase 3A protein is bound, thereby converting the substrate peptide into a reaction product; an approach step of contacting the donor bead to which the reaction product is bound with a luminescent or excitable acceptor bead to which a substance having a binding ability to the reaction product is bound, thereby bringing the donor bead and the acceptor bead into close proximity; and a detection step of detecting light generated from the donor bead or the acceptor bead due to the approach of the donor bead and the acceptor bead, one of which is excitable and the other is luminescent.

[0110] The substrate peptide of the lysine demethylase 3A protein is not limited to, and may be, for example, (i) a full-length polypeptide of a protein that serves as a substrate for the lysine demethylase 3A protein, (ii) a partial polypeptide of a protein that serves as a substrate for the lysine demethylase 3A protein, or (iii) an artificially produced polypeptide, (iv) an artificially produced substrate including (i) to (iii), or (v) any combination of (i) to (iv).

[0111] The donor beads can be excitable or luminescent.

[0112] When the donor beads are excitable, the donor beads may, for example, absorb light of a specific wavelength (e.g., 520 to 620 nm) and excite a specific enzyme (e.g., a luminescent enzyme) present in the acceptor beads.

[0113] When the donor bead is luminescent, the donor bead may contain, for example, an enzyme (e.g., a luminescent enzyme) that is excited by an acceptor bead that absorbs light of a particular wavelength (e.g., 520 to 620 nm).

[0114] The substrate peptide and the donor beads are bound to each other. The manner of binding is not limited, and for example, biotin may be bound to one side and streptavidin to the other side, and the substrate peptide of the lysine demethylase 3A protein may be bound to the donor beads by the interaction between biotin and streptavidin.

[0115] In the conversion step, the lysine demethylase 3A protein comes into contact with the donor beads, thereby converting the substrate peptide bound to the donor beads into a reaction product bound to the donor beads.

[0116] The substance capable of binding to the reaction product is not limited, and may be, for example, an antibody. The antibody may be a polyclonal antibody or a monoclonal antibody. For example, commercially available antibodies may be used.

[0117] The substance capable of binding to the reaction product is bound to the acceptor beads. The manner of binding is not limited, and for example, protein A or protein G may be bound to the acceptor beads, and the substance capable of binding to the reaction product may be bound to the acceptor beads by interaction between the acceptor beads and the antibody.

[0118] The acceptor beads can be luminescent or excitable.

[0119] When the acceptor bead is luminescent, the acceptor bead may contain, for example, an enzyme (e.g., a luminescent enzyme) that is excited by a donor bead that absorbs light of a particular wavelength (e.g., 520 to 620 nm).

[0120] When the acceptor bead is excitable, the acceptor bead may, for example, absorb light of a specific wavelength (e.g., 520 to 620 nm) and excite a specific enzyme (e.g., a luminescent enzyme) present in the donor bead.

[0121] In the approaching step, the donor bead to which the reaction product is bound is brought into contact with the acceptor bead to which a substance capable of binding to the reaction product is bound, thereby binding the reaction product to the substance capable of binding to the reaction product, and as a result, the donor bead and the acceptor bead are brought into close proximity to each other.

[0122] The donor bead and the acceptor bead are such that one is excitable and the other is luminescent, such that when the donor bead and the acceptor bead are brought into close proximity, the excitable bead activates the luminescent bead, causing it to emit light (e.g., fluorescence).

[0123] In the detection step, light generated from the donor beads or the acceptor beads is detected. At this time, strong light detected indicates that a large amount of reaction product is produced, that is, the activity of the lysine demethylase 3A protein is high. On the other hand, weak light detected at this time indicates that a small amount of reaction product is produced, that is, the activity of the lysine demethylase 3A protein is low.

[0124] In the determination step, the activity of the lysine demethylase 3A protein measured in the measurement step is compared with a control activity to determine whether or not the candidate substance is a therapeutic or preventive agent for abnormal angiogenesis.

[0125] The control activity can be, for example, the demethylation activity exhibited by the lysine demethylase 3A protein after contact with a known activity inhibitor of the lysine demethylase 3A protein, and / or the demethylation activity exhibited by the lysine demethylase 3A protein that has not been contacted with a known activity inhibitor of the lysine demethylase 3A protein.

[0126] The activity (demethylation activity) value of the lysine demethylase 3A protein measured in the above measurement step is designated as "A", the demethylation activity value exhibited by the lysine demethylase 3A protein after contact with a known activity inhibitor of the lysine demethylase 3A protein (e.g., 2,4-pyridinedicarboxylic acid) is designated as "B", and the demethylation activity value exhibited by the lysine demethylase 3A protein that has not been contacted with a known activity inhibitor of the lysine demethylase 3A protein is designated as "C".

[0127] If the value of A is approximately the same as the value of B, the candidate substance used in the contacting step can be determined to be a therapeutic or preventive agent for abnormal angiogenesis. More specifically, if the value of "A / B" is 0.8 to 1.2, 0.9 to 1.1, or approximately 1.0, the candidate substance used in the contacting step can be determined to be a therapeutic or preventive agent for abnormal angiogenesis.

[0128] If the value of A is approximately the same as the value of C, it can be determined that the candidate substance used in the contacting step is not a therapeutic or preventive agent for abnormal angiogenesis. More specifically, if the value of "A / C" is 0.8 to 1.2, 0.9 to 1.1, or approximately 1.0, it can be determined that the candidate substance used in the contacting step is not a therapeutic or preventive agent for abnormal angiogenesis.

[0129] It is preferable to have an administration step of administering the candidate substance to an abnormal angiogenesis model animal after the determination step. With this configuration, a therapeutic or preventive agent for abnormal angiogenesis can be screened more accurately.

[0130] The abnormal angiogenesis model animal is not limited, and examples thereof include an OIR model mouse, an RVO (Retinal Vein Occlusion) model mouse, and an RVO model rabbit.

[0131] Examples of the OIR model mouse that can be used include mice described in Kip M Connor et al., "Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis", Nat Protoc., 2009, 4(11), 1565-1573 and Andreas Stabl et al., "The Mouse Retina as an Angiogenesis Model", Investigative Ophthalmology and Visual Science, June 2010, Vol. 51, No. 6, 2813-2826.

[0132] As the RVO model mouse, for example, the mouse described in "Shinichiro Fuma et al., "A pharmacological approach in newly established retinal vein occlusion model" Scientific Reports, 2017 Mar 2, 7:43509" can be used.

[0133] As the RVO model rabbit, for example, the mouse described in "Takuma Neo et al., "Gene expression profile analysis of the rabbit retinal vein occlusion model" PLOS ONE, July 31, 2020, 15(7), e0236928" can be used. EXAMPLES

[0134] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0135] <1. Screening of Lysine Demethylase 3A Inhibitors> According to click chemistry, various azide compounds were reacted with various alkyne compounds in the presence of a copper catalyst to synthesize various azide-alkyne conjugates (see also the above-mentioned synthetic scheme).

[0136] Below, synthesis examples of compounds used in click chemistry are described.

[0137] Ethyl 2-(phenylamino)nicotinate Aniline (750 mg, 8.06 mmol) was added to an ethylene glycol (16 mL) solution containing ethyl 2-chloronicotinate (1.48 g, 8.00 mmol), and the mixture was heated at 120° C. for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, water (15 mL) was added, and the mixture was extracted with ethyl acetate. The obtained organic phase was washed with brine and then dried with Na2SO4. After the solvent was distilled off from the organic phase, the obtained residue was purified by column chromatography (hexane / AcOEt=9 / 1) to obtain 1.90 g of a white solid. The yield was 98%. The analytical data of the white solid are shown below.

[0138] 1 H NMR (400 MHz, CDCl3, δ; ppm) 10.2 (1H, s), 8.37 (1H, dd, J = 4.6 and 2.0 Hz), 8.25 (1H, dd, J = 7.6 and 2.0 Hz), 7.70 (2H, d, J = 8.0 Hz), 7.34 (2H, dd, J = 8.0, 7.2 Hz), 7.05 (1H, t, J = 7.2 Hz), 6.71 (1H, dd, J = 7.6, 4.6 Hz), 4.39 (2H, q, J = 7.1 Hz), 1.42 (3H, t, J = 7.1 Hz); 13C NMR (100 MHz, CDCl3, δ; ppm) 167.5, 156.2, 153.1, 140.1, 139.7, 128.8, 122.8, 120.9, 113.2, 107.2, 61.2, 14.3.

[0139] (2-(Phenylamino)pyridin-3-yl)methanol A THF (20 mL) solution containing LiAlH4 (265 mg, 6.99 mmol) was cooled to 0°C, and then a THF (15 mL) solution containing ethyl 2-(phenylamino)nicotinate (1.70 g, 7.00 mmol) was slowly added dropwise to the THF solution. The mixture was warmed to room temperature and stirred for 2 hours. After the reaction was completed for 2 hours, the mixture was cooled to 0°C, and water (0.2 mL), 15% NaOH aqueous solution (0.2 mL), and water (0.6 mL) were added to the mixture in that order, and the mixture was stirred for 30 minutes to stop the reaction. The reaction suspension was filtered through Celite, and the filtrate was concentrated. The resulting residue was purified by column chromatography (hexane / AcOEt=6 / 4 changed to 5:5) to obtain 1.36 g of a pale yellow oily product. The yield was 97%. The analytical data of the pale yellow oily product are shown below.

[0140] 1 H NMR (400 MHz, CDCl3, δ; ppm) 8.18 (1H, d, J = 5.2 Hz), 7.69 (1H, brs), 7.55 (2H, d, J = 3.6 Hz), 7.35-7.29 (3H, m), 6.99 (1H, t, J = 7.2 Hz), 6.70 (1H, dd, J = 7.6, 5.2 Hz), 4.68 (2H, s), 1.87 (1H, brs); 13 C NMR (100 MHz, CDCl3, δ; ppm) 155.0, 147.5, 140.6, 136.6, 128.9, 121.9, 120.0, 119.5, 114.4, 64.0.

[0141] 3-(Azidomethyl)-N-phenylpyridin-2-amine SOCl2 (0.550 mL, 7.58 mmol) was slowly added to a solution of (2-(phenylamino)pyridin-3-yl)methanol (1.00 g, 5.00 mmol) in CH2Cl2 (20 mL), and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was concentrated to obtain 1.21 g of a pale yellow solid. The obtained pale yellow solid was used as it was in the next reaction.

[0142] NaN3 (651 mg, 10.0 mmol) was added to a DMF solution (20 mL) containing the crude product (1.21 g), and the mixture was heated at 90°C for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, water (15 mL) was added, and the mixture was extracted with ethyl acetate. The obtained organic phase was washed with brine and then dried with Na2SO4. After the solvent was distilled off from the organic phase, the obtained residue was purified by column chromatography (hexane / AcOEt=19 / 1 changed to 10 / 1) to obtain 889 mg of a yellow solid. The yield was 79% after two steps. The analytical data of the yellow solid are shown below.

[0143] 1 H NMR (400 MHz, CDCl3, δ; ppm) 8.25 (1H, d, J = 5.2 Hz), 7.53 (1H, d, J = 8.0 Hz), 7.45 (2H, d, J = 7.6 Hz), 7.33 (2H, dd, J = 8.0, 7.2 Hz, 2H), 7.03 (1H, t, J = 7.2 Hz), 6.78 (1H, brs), 6.78 (1H, dd, J = 7.6, 5.2 Hz), 4.34 (2H, s); 13 C NMR (100 MHz, CDCl3, δ; ppm) 154.2, 148.3, 140.2, 138.1, 128.9, 122.4, 119.9, 115.6, 114.9, 52.8; HRMS (ESI) m / z: [M+H] +Calcd for C 12 H 12 N5226.1087; Found 226.1086.

[0144] Methyl 6-bromonicotinate SOCl2 (1.10 mL, 15.2 mmol) was slowly added to a toluene (20 mL) solution containing 6-Bromonicotinic acid (2.02 g, 10.0 mmol) and DMF (40 μL, 0.514 mmol), and the mixture was heated at 40° C. for 2 hours. After the reaction was completed, the mixture was concentrated to obtain 2.35 g of a pale yellow solid. The obtained pale yellow solid was used as it was in the next reaction.

[0145] Et3N (2.75 mL, 19.8 mmol) was added to a solution of the crude product (2.35 g) in MeOH (20 mL), and the mixture was heated at 40° C. for 2 hours. After the reaction was completed, the mixture was cooled to room temperature, water (15 mL) was added, and the mixture was extracted with ethyl acetate. The obtained organic phase was washed with brine and then dried with Na2SO4. After the solvent was distilled off from the organic phase, the obtained residue was purified by column chromatography (hexane / AcOEt=19 / 1 changed to 10 / 1) to obtain 2.08 g of a white solid. The yield was 94% after two steps. The analytical data of the white solid are shown below.

[0146] 1 H NMR (400 MHz, CDCl3, δ; ppm) 9.00 (1H, d, J = 1.6 Hz), 8.25 (1H, dd, J = 8.4, 1.6 Hz), 7.42 (1H, d, J = 8.4 Hz), 3.96 (3H, s); 13 C NMR (100 MHz, CDCl3) 164.9, 155.7, 151.2, 139.6, 125.0, 124.2, 52.6.

[0147] Methyl 6-((trimethylsilyl)ethynyl)nicotinate Trimethylsilylacetylene (1.05mL, 7.59mmol) was added to a MeCN (20mL) solution containing methyl 6-romonicotinate (1.08g, 5.00mmol), PdCl2(PPh3)2 (176mg, 0.251mmol), CuI (48.0mg, 0.252mmol), and Et3N (1.40mL, 10.1mmol) under an argon atmosphere. The mixture was heated at 50°C for 12 hours under an argon atmosphere. After the reaction was completed, diethyl ether was added to the residue obtained by distilling off the solvent from the mixture, and the solid was removed from the diethyl ether solution by filtration. The filtrate was concentrated, and the residue was purified by column chromatography (hexane / AcOEt=50 / 1 to 20 / 1) to obtain 1.08g of a pale yellow solid. The yield was 92%. The analytical data of the pale yellow solid is shown below.

[0148] 1 H NMR (400 MHz, CDCl3, δ; ppm) 9.15 (1H, d, J = 1.6 Hz), 8.24 (1H, dd, J = 8.2, 2.4 Hz), 7.52 (1H, dd, J = 8.2, 1.6 Hz), 3.95 (3H, s), 0.28 (9H, s); 13 C NMR (100 MHz, CDCl3) 165.3, 151.0, 146.6, 137.1, 126.7, 124.8, 103.0, 98.5, 52.5, -0.42.

[0149] Methyl 6-ethynylnicotinate K2CO3 (554 mg, 4.01 mmol) was added to a CH2Cl2 / MeOH (10 mL / 5.0 mL) solution containing methyl 6-((trimethylsilyl)ethynyl)nicotinate (467 mg, 2.00 mmol), and the mixture was stirred at room temperature for 5 hours. After the reaction was completed, water (10 mL) was added to the mixture, the mixture was extracted with CH2Cl2, and the resulting organic phase was washed with brine and then dried with Na2SO4. After the solvent was distilled off from the organic phase, the resulting residue was purified by column chromatography (hexane / AcOEt=10 / 1) to obtain 297 mg of a white solid. The yield was 92%. The analytical data of the white solid are shown below.

[0150] 1 H NMR (400 MHz, CDCl3, δ; ppm) 9.17 (1H, d, J = 1.6 Hz), 8.27 (1H, dd, J = 7.6, 1.6 Hz), 7.55 (1H, dd, J = 8.0, 1.2 Hz), 3.96 (3H, s), 3.31 (1H, s); 13 C NMR (100 MHz, CDCl3) 165.1, 151.0, 145.8, 137.3, 127.0, 125.3, 82.2, 80.0, 52.6.

[0151] Methyl 6-(1-((2-(phenylamino)pyridin-3-yl)methyl)-1H-1,2,3-triazol-4-yl)nicotinate(TAB-047) CuSO4·5H2O (13.1mg, 0.0525mmol) and sodium L-ascorbate (30.4mg, 0.154mmol) were added to a THF / H2O (5.0mL / 5.0mL) solution containing azide (112.4mg, 0.500mmol) and alkyne (84.9mg, 0.527mmol), and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the mixture was extracted with ethyl acetate, and the obtained organic phase was washed with brine and then dried over Na2SO4. After distilling off the solvent from the organic phase, the obtained residue was purified by column chromatography (hexane / AcOEt=2 / 1 changed to 0 / 1) to obtain 168mg of a white solid. The yield was 87%. The analytical data of the white solid are shown below.

[0152] 1 H NMR (400 MHz, CDCl3, δ; ppm) 9.15 (1H, d, J = 2.0 Hz), 8.38 (1H, dd, J = 8.4, 2.0 Hz), 8.78 (3H, m), 7.60-7.58 (2H, m), 7.54 (2H, d, J = 7.2 Hz), 7.32 (2H, dd, J = 7.4, 7.2 Hz), 7.02 (1H, t, J = 7.4 Hz), 6.81 (1H, dd, J = 7.2, 5.2 Hz), 5.57 (2H, s), 3.96 (3H, s); 13 C NMR (100 MHz, CDCl3, δ; ppm) 165.5, 154.4, 153.1, 150.9, 149.3, 148.5, 140.2, 138.8, 138.1, 128.9, 125.1, 123.3, 122.6, 120.1, 119.6, 115.1, 114.9, 52.4, 51.8; HRMS (ESI) m / z: [M+H] + calcd for C 21 H 19 N6O2387.1564; found 387.1561.

[0153] 6-(1-((2-(phenylamino)pyridin-3-yl)methyl)-1H-1,2,3-triazol-4-yl)nicotinic acid (TAB-050) LiOH·H2O (16.6 mg, 0.396 mmol) was added to a THF / H2O (5.0 mL / 5.0 mL) solution containing TAB-047 (116.2 mg, 0.301 mmol), and the mixture was stirred at room temperature for 10 hours. After the reaction was completed, 2 M hydrochloric acid was added to the mixture to adjust the pH to 3, and the mixture was extracted with ethyl acetate. The resulting organic phase was washed with brine and then dried over Na2SO4. The solvent was distilled off from the organic phase to obtain 80.4 mg of a white solid. The yield was 72%. The analytical data of the white solid are shown below.

[0154] 1 H NMR (400 MHz, DMSO-d6, δ; ppm) 9.06 (1H, d, J = 1.2 Hz), 8.81 (1H, s), 8.36-8.33 (2H, m), 8.16-8.12 (2H, m), 7.59 (2H, d, J = 7.2 Hz), 7.38 (2H, dd, J = 7.2, 1.6 Hz), 7.27 (2H, dd, J = 7.6, 7.2 Hz), 6.92 (1H, t, J = 7.6 Hz), 6.82 (1H, dd, J = 7.6, 4.7 Hz), 5.83 (2H, s); 13 C NMR (100 MHz, DMSO-d6, δ; ppm) 165.9, 153.1, 153.0, 150.6, 147.3, 146.6, 141.1, 138.2, 137.8, 128.3, 125.4, 124.7, 121.3, 119.9, 119.1, 116.8, 115.1, 49.3; HRMS (ESI) m / z: [M+H] + calcd for C 20 H 17 N6O2373.1408; found 373.1404.

[0155] The azide-alkyne conjugates prepared as described above were used as a library for screening lysine demethylase 3A inhibitors. The library contained the following two compounds.

[0156] [ka]

[0157] The synthesized azide-alkyne conjugate was subjected to AlphaScreen Assay without purification to evaluate the inhibitory activity of the azide-alkyne conjugate against lysine demethylase 3A. In the AlphaScreen Assay, a recombinant enzyme manufactured by BPS Biosciences, and acceptor beads and donor beads manufactured by PerkinElmer were used to evaluate the inhibitory activity against lysine demethylase 3A. The outline of the AlphaScreen Assay is described below.

[0158] First, 2.5 μL of an assay buffer solution (3% DMSO) containing 100 μM of a sample (azide-alkyne conjugate) was added to a well of a white opaque OptiPlate™-384 to serve as a control or blank.

[0159] Then, 5.0 μL of enzyme solution (lysine demethylase 3A protein in assay buffer) was added to each well of the test plate (for blanks, 5.0 μL of assay buffer was added instead of enzyme solution) and 2.5 μL of the mixture (substrate peptide / 2-OG (50 μM) / Fe (5 μM) / Asc (100 μM)). The final concentration of the enzyme was 0.2 nM and the final concentration of the substrate peptide was 60 nM. The test plate was incubated for 2 hours at room temperature with gentle shaking (250 rpm).

[0160] Then, 5.0 μL (100 μg / mL) of epigenetic buffer containing antibody-bound acceptor beads was added to each well. The test plate was incubated for 1 hour at room temperature with gentle shaking (250 rpm).

[0161] Then, 10 μL of epigenetic buffer with donor beads (50 μg / mL) was added to each well. The test plate was incubated for 30 minutes at room temperature in the dark.

[0162] The alpha signal generated in each well was measured using an Ensight™ multilabel reader (PerkinElmer Ltd.) under conditions of an excitation wavelength of 615 nm and an emission wavelength of 655 nm.

[0163] Enzyme activity A was calculated from the Alpha signal readings from the wells containing the sample (azide-alkyne conjugate). Enzyme activity B was calculated from the Alpha signal readings from the control wells. The ratio of enzyme activity A to enzyme activity B, which was set to 100%, was calculated.

[0164] 101 and 102 in Figure 1 show the results of evaluating the inhibitory activity of the azide-alkyne conjugate against lysine demethylase 3A. In 101 and 102 in Figure 1, "CTRL" indicates the enzyme activity of lysine demethylase 3A in the absence of a compound having inhibitory activity. As is clear from 101 and 102 in Figure 1, both TAB047 and TAB050 were found to have inhibitory activity against lysine demethylase 3A.

[0165] Intravitreal administration of TAB047 and TAB050 In OIR model mice with abnormal angiogenesis (see Kip M Connor et al., "Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis" Nat Protoc., 2009, 4(11), 1565-1573 and Andreas Stabl et al., "The Mouse Retina as an Angiogenesis Model" Investigative Ophthalmology and Visual Science, June 2010, Vol.51, No.6, 2813-2826), TAB047 hydrochloride or TAB050 hydrochloride was administered intravitreally to examine the effect on abnormal angiogenesis.

[0166] Seven-day-old (P7) mice were reared under 75% oxygen by volume for 5 days until they reached 12-day-old (P12) age. The 12-day-old (P12) mice were then reared under atmospheric conditions. By switching rearing conditions from high oxygen concentration to low oxygen concentration, abnormal angiogenesis (formation of ischemic areas (in other words, formation of areas where blood vessels are not formed) and formation of abnormal blood vessels (in other words, formation of blood vessels running in a disorderly manner)) was induced in the retina.

[0167] The above mice were administered TAB047 hydrochloride (1 mM) or TAB050 hydrochloride (1 mM or 10 mM) into the vitreous of the right eye once at postnatal day 12 (P12), and PBS into the vitreous of the left eye once at postnatal day 12 (P12) as a control.

[0168] At 17 days of age, retinas were harvested from the mice and abnormal neovascularization on the retinas was observed under a microscope. Based on the images, the area of ​​the entire retina, the area of ​​the ischemic area, and the area of ​​the abnormal vascular area were calculated.

[0169] Figure 2, 201 and 202, show the test results for the hydrochloride salt of TAB047.

[0170] The image of the retina is shown in 201 of Figure 2. As is clear from the image, in the control mice, the formation of ischemic areas and abnormal blood vessels was observed over a wide area of ​​the retina. On the other hand, in the mice administered TAB047 hydrochloride, the formation of ischemic areas and abnormal blood vessels was observed only over a narrow area of ​​the retina.

[0171] 202 in FIG. 2 shows a graph of the ratio of the area of ​​the abnormal vascular region to the total area of ​​the retina, and the ratio of the area of ​​the ischemic region to the total area of ​​the retina. As is clear from the graph, in the control mice, the formation of ischemic regions and abnormal blood vessels was observed over a wide area of ​​the retina. On the other hand, in the mice administered with TAB047 hydrochloride, the formation of ischemic regions and abnormal blood vessels was observed only over a narrow area of ​​the retina.

[0172] Figure 3, 301 to 303, shows the test results of the hydrochloride salt of TAB050.

[0173] The image of the retina is shown in FIG. 3, 301. As is clear from the image, in the control mice, the formation of ischemic areas and abnormal blood vessels was observed over a wide area of ​​the retina. On the other hand, in the mice administered TAB050 hydrochloride, the formation of ischemic areas and abnormal blood vessels was observed only over a narrow area of ​​the retina. The effect of TAB050 hydrochloride was concentration-dependent.

[0174] Graphs 302 and 303 in FIG. 3 show the ratio of the area of ​​abnormal vascular regions to the total area of ​​the retina, and the ratio of the area of ​​ischemic regions to the total area of ​​the retina. As is clear from the graphs, in the control mice, the formation of ischemic regions and abnormal blood vessels was observed over a wide area of ​​the retina. On the other hand, in the mice administered with TAB050 hydrochloride, the formation of ischemic regions and abnormal blood vessels was observed only over a narrow area of ​​the retina. The effect of TAB050 hydrochloride was concentration-dependent.

[0175] 3. Metabolic stability test of TAB047 and TAB050 The metabolic stability of TAB047, TAB047 hydrochloride, TAB050, and TAB050 hydrochloride was tested according to the method described in ACS Med. Chem. Lett. 2022 Aug 22; 13(10) 1582-1590.

[0176] TAB047, TAB047 hydrochloride, TAB050, and TAB050 hydrochloride (500 μM each) were mixed with liver microsomes (Sekisui Xeno Tech, LLC, Kansas City, KS, USA, final concentration 0.2 mg protein / mL), coenzyme group (1.3 mM NADPH, 3.3 mM G-6-P: Sigma, Marlborough, MA, USA, 3.3 mM MgCl2: Wako, Osaka, Japan), and 0.45 U / mL G6PDH (Oriental Yeast, Tokyo, Japan) for 0 to 60 minutes at 37°C. Two types of liver microsomes were used: human liver microsomes and mouse liver microsomes.

[0177] The mixtures were sampled at 0, 10, and 60 minutes after mixing. Acetonitrile was added to each mixture, which was then vortexed and centrifuged at 3500 rpm for 20 minutes.

[0178] After centrifugation, the supernatant was collected and subjected to LC-MS / MS to measure the amounts of TAB047, TAB047 hydrochloride, TAB050, and TAB050 hydrochloride present in the supernatant.

[0179] The ratio of the amount of TAB047, TAB047 hydrochloride, TAB050, and TAB050 hydrochloride present in the supernatant 10 minutes and 60 minutes after mixing to the amount of TAB047, TAB047 hydrochloride, TAB050, and TAB050 hydrochloride present in the supernatant 0 minutes after mixing (100%) was calculated.

[0180] The test results for TAB047 are shown in 401 of Figure 4. In 401 of Figure 4, open circles indicate the proportion of TAB047, and filled circles indicate the proportion of TAB050. As is clear from 401 of Figure 4, TAB047 was metabolized and the amount decreased over time, while the amount of TAB050 increased over time. This indicates that TAB047 was metabolized and converted to TAB050.

[0181] The test results for the hydrochloride of TAB047 are shown in 402 of Figure 4. In 402 of Figure 4, the open circles indicate the proportion of the hydrochloride of TAB047, and the closed circles indicate the proportion of the hydrochloride of TAB050. As is clear from 402 of Figure 4, the hydrochloride of TAB047 was metabolized and the amount decreased over time, while the amount of the hydrochloride of TAB050 increased over time. This indicates that the hydrochloride of TAB047 was metabolized and converted to the hydrochloride of TAB050.

[0182] The test results for TAB050 are shown in 501 of Figure 5. In 501 of Figure 5, the black circles indicate the proportion of TAB050. As is clear from 501 of Figure 5, the amount of TAB050 did not change over time. This indicates that TAB050 is stable in vivo.

[0183] 502 in Figure 5 shows the test results for the hydrochloride of TAB050. In 502 in Figure 5, the black circles indicate the proportion of the hydrochloride of TAB050. As is clear from 502 in Figure 5, the amount of the hydrochloride of TAB050 did not change over time. This indicates that the hydrochloride of TAB050 is stable in vivo.

[0184] <4.Membrane permeability test of TAB047 and TAB050> The membrane permeability (in other words, bioabsorption) of the hydrochloride salts of TAB047 and TAB050 was tested according to the Parallel Artificial Membrane Permeation Assay (PAMPA).

[0185] The parallel artificial membrane permeability test was performed using a Falcon Genetest precoated PAMPA plate system manufactured by Corning. The specific test method was performed according to the protocol attached to the system. 2 The initial concentrations of TAB047 hydrochloride and TAB050 hydrochloride were set at 100 μM.

[0186] The test results are shown in the table below. As is clear from the table below, the membrane permeability of the hydrochloride of TAB047 was high. On the other hand, the membrane permeability of the hydrochloride of TAB050 was lower than that of the hydrochloride of TAB047, being approximately 1 / 17 of that of the hydrochloride of TAB047. This indicates that the hydrochloride of TAB047 is more easily absorbed in the body than the hydrochloride of TAB050.

[0187] [Table 1] [Industrial Applicability]

[0188] The present invention can be utilized in the treatment or prevention of abnormal angiogenesis.

Claims

1. A therapeutic or preventive agent for abnormal angiogenesis, comprising a lysine demethylase 3A inhibitor as an active ingredient.

2. The agent for treating or preventing abnormal angiogenesis according to claim 1, wherein the lysine demethylase 3A inhibitor is a compound represented by the following chemical formula 1, a derivative thereof, or a salt thereof: 【Chemistry 1】 [In formula 1, R is hydrogen or any organic group].

3. 2. The therapeutic or preventive agent for abnormal angiogenesis according to claim 1, wherein R is hydrogen, an alkyl group, an aryl group, a heterocyclic group, a fluoroalkyl group, an alkenyl group, a fluoroalkenyl group, an alkynyl group, a fluoroalkynyl group, an alkoxyl group, a fluoroalkoxyl group, an acetyl group, a carboxyalkyl group, or an alkylamide group.

4. The method of claim 1, wherein the abnormal angiogenesis is the formation of an ischemic area and / or the formation of abnormal blood vessels.

5. The therapeutic or preventive agent for abnormal angiogenesis according to any one of claims 1 to 4, wherein the abnormal angiogenesis is abnormal angiogenesis associated with retinal ischemic disease, cancer, retinopathy of prematurity, age-related macular degeneration, diabetic macular edema, pathological myopia, macular edema associated with retinal vein occlusion, or neovascular glaucoma.

6. Use of a lysine demethylase 3A inhibitor for the manufacture of an agent for treating or preventing abnormal angiogenesis.

7. A compound represented by the following chemical formula 1, a derivative thereof, or a salt thereof: 【Chemistry 2】 [In formula 1, R is hydrogen or any organic group].

8. The compound, derivative, or salt thereof according to claim 7, wherein R is hydrogen, an alkyl group, an aryl group, a heterocyclic group, a fluoroalkyl group, an alkenyl group, a fluoroalkenyl group, an alkynyl group, a fluoroalkynyl group, an alkoxyl group, a fluoroalkoxyl group, an acetyl group, a carboxyalkyl group, or an alkylamide group.

9. a contacting step of contacting the lysine demethylase 3A protein with a candidate substance for a therapeutic or preventive agent for abnormal angiogenesis; a measuring step of measuring the activity of the lysine demethylase 3A protein after the contacting step; A screening method for a therapeutic or preventive agent for abnormal angiogenesis, comprising a determination step of determining whether the candidate substance is a therapeutic or preventive agent for abnormal angiogenesis by comparing the activity of the lysine demethylase 3A protein measured in the measurement step with a control activity.

10. The measuring step includes: a conversion step of contacting the lysine demethylase 3A protein with an excitable or luminescent donor bead to which a substrate peptide of the lysine demethylase 3A protein is bound, thereby converting the substrate peptide into a reaction product; an approaching step of bringing the donor bead to which the reaction product is bound into contact with a luminescent or excitable acceptor bead to which a substance capable of binding to the reaction product is bound, thereby bringing the donor bead and the acceptor bead into close proximity; 10. The method for screening therapeutic or preventive agents for abnormal angiogenesis as described in claim 9, further comprising a detection step of detecting light emitted from the donor bead or the acceptor bead upon approaching each other, wherein one of the donor bead and the acceptor bead is excitable and the other is luminescent, and detecting the light emitted from the donor bead or the acceptor bead upon approaching each other.

11. 10. The method for screening an agent for treating or preventing abnormal angiogenesis according to claim 9, further comprising, after the determination step, an administration step of administering the candidate substance to an abnormal angiogenesis model animal.

12. The method for screening a therapeutic or preventive agent for abnormal angiogenesis according to claim 11, wherein the abnormal angiogenesis model animal is an OIR model mouse, an RVO (Retinal Vein Occlusion) model mouse, or an RVO model rabbit.

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