Antibacterial compound

Abstract

To provide a compound that can be readily synthesized, exhibits antibacterial activity against Porphyromonas gingivalis being a pathogenic bacterium of periodontitis, with greater specificity, and exhibits increased stability against esterase as well as reduced cytotoxicity.SOLUTION: The present invention provides a compound represented by a general formula (1) [where R1 is a halogen atom or a nitro group, and R2 is an alkoxy group or an acylamino group] or a salt thereof or a solvate thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an antibacterial dipeptide compound and the like.

Background Art

[0002] Porphyromonas gingivalis, a causative bacterium of periodontal disease, is a non-sugar-fermenting Gram-negative bacterium that uses amino acids obtained by decomposing proteins and peptides with the bacterium-specific dipeptidyl peptidase (DPP) instead of carbohydrates as a nutrient source. It has been reported that Porphyromonas gingivalis lacking all DPP genes has a significantly reduced growth rate, and bacterial DPP inhibitors are expected as antibacterial agents based on a new mechanism of action. Patent Document 1 reports that a dipeptide compound having a specific structure exhibits bacterial DPP7 inhibitory activity.

[0003] Periodontal disease is the most prevalent bacterial infectious disease in the world and causes tooth loss. It has been clarified that periodontal disease is associated with various systemic diseases such as arteriosclerosis and diabetes, and control of periodontal disease can contribute to extending the healthy life span. Antibacterial drugs are used for the treatment of periodontal disease, but dealing with the increase in drug-resistant bacteria is an urgent issue. In addition to reducing the use of existing antibacterial drugs, the development of new narrow-spectrum antibacterial drugs is desired. However, there has been no antibacterial drug specific to periodontal pathogenic bacteria so far, and its development is an urgent need.

[0004] Instead of existing antibacterial drugs, natural antibacterial peptides produced by lactic acid bacteria have attracted attention. However, generally, these amino acid sequences are as long as 10 to 35, so high technology and costs are required for chemical synthesis, which is a problem. That is, the development of an antibacterial agent that specifically acts on periodontal pathogenic bacteria, has low cost, and ensures safety is desired.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] The present invention aims to provide a compound that can be easily synthesized, exhibits more specific antibacterial activity against Porphyromonas gingivalis, a causative agent of periodontal disease, has higher stability against esterases, and has lower cytotoxicity. [Means for solving the problem]

[0007] In view of the above problems, the inventors diligently conducted research and found that the above problems can be solved if the dipeptide compound has a structure in which the terminal carboxyl group of phenylalanine is isopropyl esterified and the 4th position of the benzene ring of the side chain is substituted with a specific group. Based on this finding, the inventors furthered their research and completed the present invention. That is, the present invention encompasses the following aspects.

[0008] Section 1. General formula (1):

[0009] [ka] [In the formula: R 1 R represents a halogen atom or a nitro group. 2 This indicates an alkoxy group or an acylamino group. A compound represented by the same, or a salt thereof, or a solvate thereof.

[0010] Section 2.R 2 The compound described in item 1, or a salt thereof, or a solvate thereof, wherein the compound is an alkoxy group having 1 to 4 carbon atoms or an acylamino group having 2 to 5 carbon atoms.

[0011] Item 3. The compounds described in Item 1, or salts thereof, or solvates thereof, wherein the halogen atom is a chlorine atom.

[0012] Section 4.R1 is a halogen atom and R 2 is an alkoxy group, or R 1 is a nitro group and R 2 is an alkoxy group or an acylamino group, the compound according to claim 1 or a salt thereof or a solvate thereof.

[0013] Item 5. R 1 is a chlorine atom and R 2 is an alkoxy group having 1 to 4 carbon atoms, or R 1 is a nitro group and R 2 is an alkoxy group having 1 to 4 carbon atoms or an acylamino group having 2 to 5 carbon atoms, the compound according to claim 1 or a salt thereof or a solvate thereof.

[0014] Item 6. An antibacterial agent containing the compound according to any one of Items 1 to 5 or a salt thereof or a solvate thereof.

[0015] Item 7. The antibacterial agent according to Item 6, wherein the target bacterium is Porphyromonas gingivalis.

[0016] Item 8. A prophylactic or ameliorating agent for bacterial infections containing the compound according to any one of Items 1 to 5 or a salt thereof or a solvate thereof.

[0017] Item 9. The prophylactic or ameliorating agent according to Item 8, wherein the bacterial infection is periodontal disease.

[0018] <00001​​​​​​​​​​​

[0020] [Figure 1] This shows the amount of residual ester in the presence of human carboxyesterase 1, as measured in Test Example 1. [Figure 2] The cell viability measured in Test Example 4 is shown. [Modes for carrying out the invention]

[0021] In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.”

[0022] 1.Compound In one embodiment, the present invention is based on the general formula (1):

[0023] [ka] This invention relates to compounds represented by [formula], their salts, or solvates thereof (in this specification, these may be collectively referred to as "compounds of the present invention"). This is described below.

[0024] R 1 This indicates a halogen atom or a nitro group.

[0025] Examples of halogen atoms include fluorine, chlorine, bromine, and iodine atoms. Among halogen atoms, chlorine atoms are particularly preferred from the viewpoint of antibacterial activity and other factors.

[0026] R 1 From the viewpoint of antibacterial activity and other factors, a nitro group is particularly preferred.

[0027] R 2 This indicates an alkoxy group or an acylamino group.

[0028] There are no particular restrictions on the alkoxy group, and examples include linear or branched (preferably linear) alkoxy groups having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms, and particularly preferably 1 carbon atom, from the viewpoint of antibacterial activity, etc. Examples of alkoxy groups include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, etc.

[0029] The acylamino group is not particularly limited as long as it is a group formed by substituting one hydrogen atom of an amino group (-NH2) with an acyl group. Examples include linear or branched (preferably linear) acylamino groups with 2 to 7 carbon atoms, preferably 2 to 5 carbon atoms, more preferably 2 to 3 carbon atoms, and even more preferably 2 carbon atoms, from the viewpoint of antibacterial activity, etc. Examples of acylamino groups include acetylamino group, propanoylamino group, butanoylamino group, pentanoylamino group, etc.

[0030] R 2 From the viewpoint of antibacterial activity and other factors, an alkoxy group is particularly preferred.

[0031] From the perspective of antibacterial activity, R 1 is a halogen atom and R 2 is an alkoxy group, or R 1 is a nitro group and R 2 It is preferable that R is an alkoxy group or an acylamino group. 1 is a nitro group and R 2 It is more preferable that R is an alkoxy group or an acylamino group. 1 is a nitro group and R 2 It is particularly preferable that the group is an alkoxy group.

[0032] Compounds represented by general formula (1) include stereoisomers and optical isomers, and these are not particularly limited. Among the compounds represented by general formula (1), compounds having a preferred stereostructure include the following general formula (1'):

[0033] [ka] [In the formula, R 1 and R 2 This is the same as above. Examples of compounds represented by [the formula shown] are given.

[0034] The salts of compounds represented by general formula (1) are not particularly limited, as long as they are pharmaceutically acceptable salts. For example, acidic salts can be used as such salts. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; and organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and p-toluenesulfonate.

[0035] The compound represented by general formula (1) or its salt can also be a solvate. Examples of solvents include water and pharmaceutically acceptable organic solvents (e.g., ethanol, glycerol, acetic acid, etc.).

[0036] The compounds of the present invention can be easily synthesized. Typically, they can be obtained by a method involving the peptide bonding of two amino acids and / or amino acid derivatives. More specifically, they can be synthesized according to or in accordance with the methods described in Patent Document 1 and the examples below.

[0037] 2.Applications The compounds of the present invention can exhibit antibacterial activity more specifically against Porphyromonas gingivalis, a bacterium that causes periodontal disease. For this reason, the compounds of the present invention can be suitably used as active ingredients in antibacterial agents (particularly antibacterial agents against Porphyromonas gingivalis), agents for the prevention or improvement (or treatment) of bacterial infections (particularly periodontal disease), etc. From this viewpoint, in one embodiment, the present invention relates to antibacterial agents, agents for the prevention or improvement of bacterial infections, etc. (in this specification, these may be collectively referred to as "agents of the present invention") that contain a compound represented by general formula (1) or a salt thereof or a solvate thereof. Furthermore, the compounds of the present invention are thought to have bacterial dipeptidyl peptidase 7 inhibitory activity.

[0038] Examples of target bacteria include sugar-non-fermenting Gram-negative bacteria such as Bacteroides fragilis, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Capnocytophaga gingivalis, Capnocytophaga ochracea, Chryseobacterium sp., Shewanella putrefaciens, Stenotrophomonas maltophilia, Porphyromonas assaccharolytia, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas uenonis, Prevotella bivia, Prevotella disiens, Prevotella intermedia, Prevotella melaninogenica, Prevotella oralis, Prevotella oris, Pseudomonas sp., Pseudoxanthomonas mexicana, and Tannerella forsythensis. Among these, Porphyromonas gingivalis is particularly preferred.

[0039] Bacterial infections are not particularly limited as long as they are caused by the bacteria mentioned above, and examples include periodontal disease, sepsis, bacteremia, and pneumonia. Among these, periodontal disease is particularly preferred.

[0040] The amount of the active ingredient in the preparation of the present invention is not particularly limited as long as it is in an amount that can exert an antibacterial effect. The amount of the active ingredient in the preparation of the present invention can be approximately 0.0001 parts by weight to 100 parts by weight, based on 100 parts by weight of the entire preparation of the present invention.

[0041] In the agent of the present invention, a compound represented by general formula (1) or a salt thereof, or a solvate thereof, may be used in combination with an inhibitor of bacterial DPP (e.g., DPP-4, -5, -7, -11, etc.). Examples of DPP inhibitors to be used in combination include anagliptin, alogliptin, omaligliptin, saxagliptin, sitagliptin, teneligliptin, trelagliptin, vildagliptin, lignagliptin, berberine, DPP4 selective inhibitor 1c, K579, NVP-DPP728, 2-cyano-1-isoleucylpyrrolidine, glycylhydroxyproline, lysinopril, etc.

[0042] The agent of the present invention may be a composition containing additives depending on the intended use and embodiment. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, rust inhibitors, metal corrosion inhibitors, defoamers, rust inhibitors, extreme pressure additives, metal corrosion inhibitors, defoamers, dyes, etc. Depending on the intended use, it is preferable to select and use pharmaceutically acceptable components and cosmetically acceptable components from among these additives.

[0043] The field of use of the agent of the present invention is not particularly limited. For example, it can be used in fields such as medicine, cosmetics, food, cleaning, oral care, and reagents.

[0044] The form of the agent of the present invention is not particularly limited and can take the form commonly used in each application, depending on the application of the agent of the present invention.

[0045] In terms of form, when the intended use is pharmaceutical, the formulations may include, for example, injectable preparations (e.g., intravenous infusion preparations, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), transdermal patches (plasters, adhesive tapes (reservoir type, matrix type, etc.), poultices, patches, microneedles, etc.), ointments, topical solutions (liniments, lotions, etc.), sprays (topical aerosols, pump sprays, etc.), creams, gels, eye drops, eye ointments, nasal drops, suppositories, semi-solid rectal preparations, enema preparations, and other formulations suitable for parenteral administration; Examples of formulations suitable for oral administration (oral formulations) include tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, and jelly-like drops), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), and jellies.

[0046] In terms of form, when the intended use is as a cosmetic, examples include liquid formulations, gel formulations, cream formulations, ointments, stick formulations, etc.

[0047] In terms of form, when the intended use is a health stimulant or nutritional supplement (such as a supplement), examples of formulations suitable for oral intake (oral formulations) include tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, and jelly-like drops), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), and jellies.

[0048] In terms of form, when used as a food composition, examples include liquid, gel-like, or solid foods such as juice, soft drinks, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, furikake (rice seasoning), infant formula, cake mix, powdered or liquid dairy products, bread, and cookies.

[0049] In terms of form, when the intended use is an oral composition (including pharmaceuticals), examples include any form such as liquid (solution, emulsion, suspension, etc.), semi-solid (gel, cream, paste, etc.), solid (tablet, particulate, capsule, film, kneaded product, molten solid, waxy solid, elastic solid, etc.), more specifically, toothpaste (toothpaste, liquid toothpaste, powder toothpaste, etc.), mouthwash, gargle, topical application, patch, oral freshener, and food (e.g., chewing gum, tablets, candy, gummies, film, lozenges, etc.).

[0050] In terms of form, when used as a disinfectant, examples include liquid formulations such as liquid preparations, emulsions, suspensions, dispersants, and aerosols; and solid or semi-solid formulations such as wettable powders, powders, granules, fine granules, and flowable formulations.

[0051] When the agent of the present invention is applied to a living organism, the route of application (e.g., administration, ingestion, etc.) is not particularly limited, and it can be administered to mammals, including humans, by any of the following routes of administration: oral administration and parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, local administration).

[0052] When the agent of the present invention is applied to a living organism, the amount applied (e.g., administration, ingestion, etc.) is not particularly limited as long as it is an effective amount that produces the desired effect, and is usually 0.1 to 1000 mg / kg body weight of the active ingredient per day. The above dosage is preferably administered once a day or divided into two to three doses, and can be increased or decreased as appropriate depending on age, disease state, and symptoms.

[0053] In one embodiment, the present invention may be a composition (for example, an oral composition, a food composition, a pharmaceutical composition, a cosmetic composition, etc.) containing a compound represented by general formula (1), a salt thereof, or a solvate thereof. The above description relating to the agent of the present invention shall be incorporated herein by reference. [Examples]

[0054] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0055] Example 1. Compound Synthesis The hydrochloride salts of the following compounds 1-7 were synthesized.

[0056] [ka]

[0057] The hydrochloride salts of compounds 1 and 2 were synthesized via the following route.

[0058] [ka]

[0059] Tert-butoxycarbonyl-(O-methyl)tyrosine was converted to an isopropyl ester with 2-bromopropane, and then the tert-butoxycarbonyl group was removed with a hydrochloric acid-ethyl acetate solution. The resulting hydrochloride salts were condensed with tert-butoxycarbonyl-4-chlorophenylalanine or tert-butoxycarbonyl-4-nitrophenylalanine by EDC, and then purified by flash chromatography. Each intermediate was deprotected to obtain the hydrochloride salts of compounds 1 and 2, which are dipeptide isopropyl esters of the present invention.

[0060] The hydrochloride salts of compounds 3-7 were synthesized via the following route.

[0061] [ka]

[0062] Tert-butoxycarbonyl-4-nitrophenylalanine was converted to an isopropyl ester with 2-bromopropane, and then the nitro group was reduced to an amino group with palladium carbon. The amino intermediate was acylated with an acid anhydride, and then the tert-butoxycarbonyl group was removed with a hydrochloric acid-ethyl acetate solution. The resulting hydrochloride salts were condensed with tert-butoxycarbonyl-4-nitrophenylalanine or tert-butoxycarbonyl-4-chlorophenylalanine by EDC, followed by deprotection to obtain the hydrochloride salts of compounds 3-7.

[0063] Example 1 - 1.4-Chlorophenylalanyl-O-methyltyrosine isopropyl ester hydrochloride (hydrochloride of compound 1) N-α-(tert-butoxycarbonyl)-O-methyl-L-tyrosine (4.43 g, 15.0 mmol) was dissolved in anhydrous N,N-dimethylformamide (100 mL), cesium carbonate (9.77 g, 30.0 mmol) was added, and the mixture was stirred at room temperature. Then 2-bromopropane (2.80 mL, 30.0 mmol) was added and the mixture was stirred at room temperature for 2 days. The reaction mixture was filtered and concentrated under reduced pressure. Ethyl acetate was added to the residue, and the mixture was washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain a colorless oil. Anisole (3.26 mL, 30.0 mmol) and 4M hydrochloric acid-ethyl acetate solution (50 mL) were added to the oil and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered and dried to obtain a white solid (yield 4.02 g, yield 97%). MS (ESI-TOF) m / z: calcd for C 13 H 20 NO3[M + H] + 238.1438; found 238.1433. The obtained O-methyltyrosine isopropyl hydrochloride (1.00 g, 3.65 mmol) was dissolved in N,N-dimethylformamide (50 mL), and under ice-cold stirring, triethylamine (510 μL, 3.65 mmol), 1-hydroxybenzotriazole monohydrate (0.614 g, 4.01 mmol), N-α-(tert-butoxycarbonyl)-4-chloro-L-phenylalanine (1.20 g, 4.01 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.769 g, 4.01 mmol) were added in sequence, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue. The mixture was then washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain a white solid (2.38 g). The resulting white solid was mixed with anisole (793 μL, 7.30 mmol) and 4M hydrochloric acid-ethyl acetate solution (12 mL), and stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered and dried. The resulting crude product was dissolved in methanol (10 mL), passed through a syringe filter (pore size 0.45 μm), concentrated under reduced pressure, and dried to obtain a white solid (yield 1.54 g, yield 92%). 1 H NMR (500 MHz, DMSO-d6) δ ppm 1.06 (d, J=6.31 Hz, 3 H), 1.16 (d, J=6.31 Hz, 3 H), 2.94 - 3.02 (m, 3 H), 3.20 - 3.24 (m, 1 H), 3.72 (s, 3 H), 4.12 (dd, J=7.57, 5.67 Hz, 1 H), 4.39 - 4.42 (m, 1 H), 4.82 - 4.86 (m, 1 H), 6.84 - 6.87 (m, 2 H), 7.18 - 7.21 (m, 2 H), 7.33 - 7.38 (m, 4 H), 8.33 (br s, 3 H), 9.30 (br d, J=7.25 Hz, 1 H); MS (ESI-TOF) m / z: calcd for C 22 H 28ClN2O4[M + H] + 419.1732; found 419.1741.

[0064] Example 1-2. 4-Nitrophenylalanyl-O-methyltyrosine isopropyl ester hydrochloride (hydrochloride of compound 2) The obtained O-methyltyrosine isopropyl hydrochloride (1.00 g, 3.65 mmol) was dissolved in N,N-dimethylformamide (50 mL), and under ice-cold stirring, triethylamine (510 μL, 3.65 mmol), 1-hydroxybenzotriazole monohydrate (0.614 g, 4.01 mmol), N-α-(tert-butoxycarbonyl)-4-nitro-L-phenylalanine (1.24 g, 4.01 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.769 g, 4.01 mmol) were added in sequence, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, ethyl acetate was added to the residue, and the mixture was washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated saline solution, and then dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain a white solid. This was then purified by flash chromatography (hexane-ethyl acetate, silica gel 40 g) and concentrated under reduced pressure to obtain a white solid (1.93 g). The obtained white solid was mixed with anisole (793 μL, 7.30 mmol) and 4M hydrochloric acid-ethyl acetate solution (12 mL) and stirred at room temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitated precipitate was filtered and dried. The resulting crude product was dissolved in methanol (10 mL), passed through a syringe filter (pore size 0.45 μm), concentrated under reduced pressure, and dried to obtain a white solid (yield 1.59 g, yield 93%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.05 (d, J=6.36 Hz, 3 H), 1.15 (d, J=6.11 Hz, 3 H), 2.95 (d, J=7.09 Hz, 2 H), 3.16 (dd, J=13.69, 7.83 Hz, 1 H), 3.33 - 3.36 (m, 1 H), 3.72 (s, 3 H), 4.21 (br t, J=6.60 Hz, 1 H), 4.41 (q, J=7.17 Hz, 1 H), 4.82 (quin, J=6.30 Hz, 1 H), 6.83 - 6.88 (m, 2 H), 7.17 - 7.24 (m, 2 H), 7.62 - 7.68 (m, 2 H), 8.10 - 8.25 (m, 2 H), 8.44 (br s, 3 H), 9.35 (d, J=7.34 Hz, 1 H); MS (ESI-TOF) m / z: calcd for C 22 H 28 N3O6[M + H] + 430.1973; found 430.1960.。

[0065] Example 1-3. 4-Nitrophenylalanyl-(4-acetylamino)phenylalanine isopropyl ester hydrochloride (hydrochloride of compound 3) N-α-(tert-butoxycarbonyl)-4-nitro-L-phenylalanine (5.00 g, 16.1 mmol) was dissolved in anhydrous N,N-dimethylformamide (50 mL), cesium carbonate (10.5 g, 32.2 mmol) was added, and under stirring at room temperature, 2-bromopropane (3.01 mL, 32.2 mmol) was added and the mixture was stirred at room temperature for 3 days. The reaction mixture was filtered and concentrated under reduced pressure. Ethyl acetate was added to the residue, and the mixture was washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain an orange solid. The solid was dissolved in isopropanol (50 mL), 10% palladium-carbon was added, and the mixture was purged with nitrogen using a balloon, followed by hydrogen purging, and vigorously stirred at room temperature for 5 hours. The reaction mixture was filtered and concentrated under reduced pressure. It was then purified by flash chromatography (hexane-ethyl acetate, silica gel 40 g) and concentrated under reduced pressure to obtain a white solid (yield 3.27 g, yield 63%). MS (ESI-TOF) m / z: calcd for C 17 H 27 N2O4[M + H] + 323.1965; found 323.1940. The obtained N-α-(tert-butoxycarbonyl)-4-aminophenylalanine isopropyl ester (500 mg, 1.55 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), and triethylamine (433 μL, 3.10 mmol) and acetyl chloride (156 μL, 2.32 mmol) were added under stirring at room temperature, and the mixture was stirred at room temperature for 1 hour. After adding ethyl acetate to the reaction mixture, it was washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain a colorless oil. 4M hydrochloric acid-ethyl acetate solution (5 mL) was added to the oil, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was then concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitated precipitate was filtered and dried to obtain a white solid (yield 437 mg, yield quant.). MS (ESI-TOF) m / z: calcd for C 14 H 21 N2O3[M + H]+ 265.1547; found 265.1526. The obtained 4-acetylaminophenylalanine isopropyl hydrochloride (30.1 mg, 0.10 mmol) was dissolved in N,N-dimethylformamide (2 mL), and under ice-cold stirring, triethylamine (14.0 μL, 0.10 mmol), 1-hydroxybenzotriazole monohydrate (16.8 mg, 0.11 mmol), N-α-(tert-butoxycarbonyl)-4-nitro-L-phenylalanine (34.1 g, 0.11 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (21.1 mg, 0.11 mmol) were added in sequence, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue. The mixture was then washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain a yellowish-white solid (42.4 mg). The obtained solid was mixed with anisole (21.7 μL, 0.20 mmol) and 4M hydrochloric acid-ethyl acetate solution (1 mL), and stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitated precipitate was filtered and dried. The resulting crude product was purified by HPLC, and after lyophilization, a white powder was obtained (yield 16.5 mg, yield 33%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.02 - 1.11 (m, 3 H), 1.11 - 1.19 (m, 3 H), 2.02 (s, 3 H), 2.89 - 3.01 (m, 2 H), 3.09 (br dd, J=13.94, 8.44 Hz, 1 H), 3.26 (br d, J=5.14 Hz, 1 H), 4.15 (br s, 1 H), 4.43 - 4.52 (m, 1 H), 4.83 (quin, J=6.27 Hz, 1 H), 7.17 (m, J=8.56 Hz, 2 H), 7.50 (m, J=8.56Hz, 2H), 7.55 - 7.62 (m, 2 H), 8.10 - 8.37 (m, 5 H), 9.06 (d, J=7.46 Hz, 1 H), 9.94 (s, 1 H); MS (ESI-TOF) m / z: calcd for C 23 H 29 N4O6[M + H] + 457.2082; found 457.2077.

[0066] Example 1-4.4-Nitrophenylalanyl-(4-propanoylamino)phenylalanine isopropyl ester hydrochloride (hydrochloride of compound 4) N-α-(tert-butoxycarbonyl)-4-aminophenylalanine isopropyl ester (150 mg, 0.46 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL), and triethylamine (130 μL, 0.93 mmol) and propanoyl chloride (60.9 μL, 0.70 mmol) were added while stirring at room temperature. The mixture was stirred at room temperature for 2 hours. Ethyl acetate was added to the reaction mixture, and the mixture was washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain a colorless oil. 4M hydrochloric acid-ethyl acetate solution (2 mL) was added to the oil, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was then concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered and dried to obtain a white solid (yield 136 mg, yield 92%). MS (ESI) m / z: calcd for C 15 H 23 N2O3[M + H]+ 279.2; found 279.2. The obtained 4-propanoylaminophenylalanine isopropyl hydrochloride (31.5 mg, 0.10 mmol) was dissolved in N,N-dimethylformamide (2 mL), and under ice-cold stirring, triethylamine (14.0 μL, 0.10 mmol), 1-hydroxybenzotriazole monohydrate (16.8 mg, 0.11 mmol), N-α-(tert-butoxycarbonyl)-4-nitro-L-phenylalanine (34.1 g, 0.11 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (21.1 mg, 0.11 mmol) were added in sequence, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue. The mixture was then washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain an orange oily substance (58.6 mg). The obtained solid was mixed with 1.5 mL of 4 M hydrochloric acid-ethyl acetate solution and stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered and dried to obtain a white solid (yield 50.6 mg, yield quant., diastereomer mixture). MS (ESI) m / z: calcd for C 24 H 31 N4O6[M + H] + 471.2; found 471.2.

[0067] Example 1-5. 4-Nitrophenylalanyl-(4-butanoylamino)phenylalanine isopropyl ester hydrochloride (hydrochloride of compound 5) N-α-(tert-butoxycarbonyl)-4-amino-L-phenylalanine isopropyl ester (150 mg, 0.46 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL), and triethylamine (130 μL, 0.93 mmol) and butanoyl chloride (72.9 μL, 0.70 mmol) were added while stirring at room temperature. The mixture was stirred at room temperature for 2 hours. Ethyl acetate was added to the reaction mixture, and the mixture was washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain a colorless oil. 4M hydrochloric acid-ethyl acetate solution (2 mL) was added to the oil, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was then concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered and dried to obtain a white solid (yield 145 mg, yield 94%). MS (ESI) m / z: calcd for C 16 H 25 N2O3[M + H] + 293.2; found 293.2. The obtained 4-butanoylaminophenylalanine isopropyl hydrochloride (32.9 mg, 0.10 mmol) was dissolved in N,N-dimethylformamide (2 mL), and under ice-cold stirring, triethylamine (14.0 μL, 0.10 mmol), 1-hydroxybenzotriazole monohydrate (16.8 mg, 0.11 mmol), N-α-(tert-butoxycarbonyl)-4-nitro-L-phenylalanine (34.1 g, 0.11 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (21.1 mg, 0.11 mmol) were added in sequence, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue. The mixture was then washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain an orange oily substance (58.2 mg). The obtained solid was mixed with 1.5 mL of 4 M hydrochloric acid-ethyl acetate solution and stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered and dried to obtain a white solid (yield 50.1 mg, yield 96%, diastereomer mixture). MS (ESI) m / z: calcd for C 25 H 33 N4O6[M + H] + 485.2; found 485.2.

[0068] Example 1-6. 4-Nitrophenylalanyl-(4-pentanoylamino)phenylalanine isopropyl ester hydrochloride (hydrochloride of compound 6) N-α-(tert-butoxycarbonyl)-4-aminophenylalanine isopropyl ester (150 mg, 0.46 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL), and triethylamine (130 μL, 0.93 mmol) and pentanoyl chloride (84.2 μL, 0.70 mmol) were added while stirring at room temperature. The mixture was stirred at room temperature for 2 hours. Ethyl acetate was added to the reaction mixture, and the mixture was washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain a colorless oil. 4M hydrochloric acid-ethyl acetate solution (2 mL) was added to the oil, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was then concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered and dried to obtain a white solid (yield 155 mg, yield 97%). MS (ESI) m / z: calcd for C 17 H 27 N2O3[M + H] + 307.2; found 307.2. The obtained 4-propanoylaminophenylalanine isopropyl hydrochloride (34.3 mg, 0.10 mmol) was dissolved in N,N-dimethylformamide (2 mL), and under ice-cold stirring, triethylamine (14.0 μL, 0.10 mmol), 1-hydroxybenzotriazole monohydrate (16.8 mg, 0.11 mmol), N-α-(tert-butoxycarbonyl)-4-nitro-L-phenylalanine (34.1 g, 0.11 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (21.1 mg, 0.11 mmol) were added in sequence, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue. The mixture was then washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated saline solution, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain an orange oily substance (59.1 mg). The obtained solid was mixed with 1.5 mL of 4 M hydrochloric acid-ethyl acetate solution and stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered and dried to obtain a white solid (yield 49.5 mg, yield 92%, diastereomer mixture). MS (ESI) m / z: calcd for C 26 H 35 N4O6[M + H] + 499.3; found 499.2.

[0069] Example 1-7. 4-Chlorophenylalanyl-(4-acetylamino)phenylalanine isopropyl ester hydrochloride (hydrochloride of compound 7) The obtained 4-acetylaminophenylalanine isopropyl hydrochloride (30.1 mg, 0.10 mmol) was dissolved in N,N-dimethylformamide (2 mL), and under ice-cold stirring, triethylamine (14.0 μL, 0.10 mmol), 1-hydroxybenzotriazole monohydrate (16.8 mg, 0.11 mmol), N-α-(tert-butoxycarbonyl)-4-chloro-L-phenylalanine (33.0 mg, 0.11 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (21.1 mg, 0.11 mmol) were added in sequence, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue. The mixture was then washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain an orange oily substance (48.0 mg). The obtained solid was mixed with 1.5 mL of 4 M hydrochloric acid-ethyl acetate solution and stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered and dried to obtain a white solid (yield 37.7 mg, yield 78%, diastereomer mixture). 1 H NMR (500 MHz, DMSO-d6) δ ppm 1.13 (d, J=6.31 Hz, 3 H), 1.19 (d, J=5.99 Hz, 3 H), 2.00 (s, 3 H), 2.69 - 2.82 (m, 2 H), 2.95 (td, J=14.58, 5.20 Hz, 2 H), 4.03 (br s, 1 H), 4.41 - 4.55 (m, 1 H), 4.88 (dt, J=12.61, 6.31 Hz, 1 H), 7.01 (d, J=8.20 Hz, 2 H), 7.13 (d, J=8.51 Hz, 2 H), 7.28 - 7.38 (m, 2 H), 7.49 (d, J=8.51 Hz, 2 H), 8.06 (br s, 3 H), 8.99 (br d, J=8.20 Hz, 1 H), 9.89 (s, 1 H); MS (ESI) m / z: calcd for C 23 H 29ClN3O4[M + H] + 446.2; found 466.3.

[0070] Comparative Example 1. Synthesis of the compound The hydrochloride salts of the following compounds (derivative 8, methyl ester of derivative 8, propyl ester of derivative 8, and methyl ester of compound 2) were synthesized.

[0071] [ka]

[0072] Comparative Example 1 - 1.3-Chlorophenylalanyl-O-methyltyrosine isopropyl ester hydrochloride (hydrochloride of derivative 8) O-methyltyrosine isopropyl hydrochloride (1.00 g, 3.65 mmol) was dissolved in N,N-dimethylformamide (50 mL), and under ice-cold stirring, triethylamine (510 μL, 3.65 mmol), 1-hydroxybenzotriazole monohydrate (0.614 g, 4.01 mmol), N-α-(tert-butoxycarbonyl)-3-chloro-L-phenylalanine (1.20 g, 4.01 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.769 g, 4.01 mmol) were added in sequence, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue. The mixture was then washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain a white solid (2.13 g). The resulting white solid was mixed with anisole (793 μL, 7.30 mmol) and 4M hydrochloric acid-ethyl acetate solution (12 mL), and stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitated product was filtered and dried. The resulting crude product was dissolved in methanol (10 mL), passed through a syringe filter (pore size 0.45 μm), concentrated under reduced pressure, and dried to obtain a white solid (yield 1.58 g, yield 95%). 1H NMR (500 MHz, DMSO-d6) δ ppm 1.06 (d, J=6.31 Hz, 3 H), 1.16 (d, J=6.31 Hz, 3 H), 2.89 - 3.05 (m, 3 H), 3.24 (dd, J=14.03, 5.20 Hz, 1 H), 3.72 (s, 3 H), 4.13 (dd, J=7.57, 5.36 Hz, 1 H), 4.41 (q, J=7.25 Hz, 1 H), 4.84 (quin, J=6.31 Hz, 1 H), 6.77 - 6.91 (m, 2 H), 7.20 (m, J=8.83 Hz, 2 H), 7.24 - 7.39 (m, 3 H), 7.44 (s, 1 H), 8.33 (br s, 3 H), 9.22 - 9.37 (m, 1 H); MS (ESI-TOF) m / z: calcd for C 19 H 29 N2O4[M + H] + 419.1732; found 419.1755.。

[0073] Comparative Example 1-2. 3-Chlorophenylalanyl-O-methyltyrosine methyl ester hydrochloride (hydrochloride salt of the methyl ester of derivative 8) O-methyltyrosine methyl ester hydrochloride (100 mg, 0.41 mmol) was dissolved in N,N-dimethylformamide (2 mL), and under ice-cold stirring, triethylamine (220 μL, 1.57 mmol), 1-hydroxybenzotriazole monohydrate (68.9 mg, 0.45 mmol), N-α-(tert-butoxycarbonyl)-3-chloro-L-phenylalanine (135 mg, 0.45 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (86.3 mg, 0.45 mmol) were added in sequence, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue. The mixture was then washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain a white solid. The obtained N-α-(tert-butoxycarbonyl)-3-chloro-L-phenylalanyl-O-methyltyrosine methyl ester was mixed with anisole (71 μL, 0.65 mmol) and 4M hydrochloric acid-ethyl acetate solution (1.1 mL), and stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitated precipitate was filtered and dried. The resulting crude product was purified by HPLC, and after lyophilization, a white powder was obtained (yield 73.5 mg, yield 42%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 2.89 - 3.03 (m, 3 H), 3.15 (dd, J=14.18, 5.01 Hz, 1 H), 3.62 (s, 3 H), 3.72 (s, 3 H), 4.08 (br. s., 1 H), 4.52 (td. 9.09 (d, J=7.58 Hz, 1 H); MS (ESI-TOF) m / z: calcd for C 20 H 24 ClN2O4[M + H] +391.1419; found, 391.1419.

[0074] Comparative Example 1-3.3-Chlorophenylalanyl-O-methyltyrosine propyl ester hydrochloride (hydrochloride of the propyl ester of derivative 8) N-α-(tert-butoxycarbonyl)-3-chloro-L-phenylalanyl-O-methyltyrosine methyl ester (444 mg, 0.903 mmol) was dissolved in methanol (3 mL), and 1 M sodium hydroxide aqueous solution (2.71 mL, 2.71 mmol) was added. The mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure to remove methanol by distillation. A 10% citric acid solution was added to the residue to make it acidic (pH 3), and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed with saturated brine and dried over sodium sulfate. The drying agent was removed by filtration, and the mixture was concentrated under reduced pressure to obtain a white solid (yield 439 mg, yield quant.). MS (ESI-TOF) m / z: calcd for C 24 H 30 ClN2O6[M + H] + 477.1787; found, 477.1760. N-α-(tert-butoxycarbonyl)-3-chloro-L-phenylalanyl-O-methyltyrosine (60 mg, 0.13 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL), and (81.5 mg, 0.25 mmol) was added. Under stirring at room temperature, 1-bromopropane (23 mg, 0.14 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was filtered and concentrated under reduced pressure. Ethyl acetate was added to the residue, and the mixture was washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the solid obtained by concentration under reduced pressure was mixed with anisole (20 μL, 0.18 mmol) and 4M hydrochloric acid-ethyl acetate solution (0.3 mL) and stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered and dried to obtain a white solid. The obtained crude product was purified by HPLC and freeze-dried to obtain a white powder (yield 34 mg, yield 58%). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.82 (t, J=7.40 Hz, 3 H), 1.47 - 1.58 (m, 2 H), 2.89 - 3.03 (m, 3 H), 3.16 (dd, J=14.18, 4.77 Hz, 1 H), 3.72 (s, 3 H), 3.92 - 4.04 (m, 2 H), 4.08 (dd, J=8.19, 5.01 Hz, 1 H), 4.47 - 4.55 (m, 1 H), 6.81 - 6.90 (m, 2 H), 7.14 - 7.21 (m, 2 H), 7.23 - 7.28 (m, 1 H), 7.31 - 7.38 (m, 2 H), 7.40 (s, 1 H), 8.16 (br. s., 3 H), 9.10 (d, J=7.58 Hz, 1 H); MS (ESI-TOF) m / z: calcd for C 22 H 28 ClN2O4[M + H] + 419.1732; found, 419.1719.。

[0075] Comparative Example 1-4.4-Nitrophenylalanyl-O-methyltyrosine methyl ester hydrochloride (hydrochloride salt of the methyl ester of compound 2) Tyrosine methyl hydrochloride (0.695 g, 3.00 mmol) was dissolved in N,N-dimethylformamide (20 mL), and under ice-cold stirring, triethylamine (419 μL, 3.00 mmol), 1-hydroxybenzotriazole (0.505 g, 3.30 mmol), N-α-(tert-butoxycarbonyl)-3-chloro-L-phenylalanine (1.02 g, 3.30 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.633 g, 3.30 mmol) were added in sequence, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue. The mixture was then washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the mixture was concentrated under reduced pressure to obtain a white solid (1.50 g). The obtained solid was dissolved in dehydrated N,N-dimethylformamide (15 mL), potassium carbonate (0.829 g, 6.00 mmol) was added, and methyl iodide (747 μL, 30.0 mmol) was added while stirring at room temperature. The mixture was stirred at room temperature for 1 day. The reaction mixture was filtered and concentrated under reduced pressure. Ethyl acetate was added to the residue, and the mixture was washed sequentially with 10% citric acid solution, 5% sodium bicarbonate aqueous solution, and saturated brine, and dried over sodium sulfate. The drying agent was filtered off, and the colorless oily substance obtained by concentration under reduced pressure was purified by flash chromatography (hexane-ethyl acetate, silica gel 40 g) and concentrated under reduced pressure to obtain a white solid (1.45 g). The obtained white solid was mixed with anisole (652 μL, 6.00 mmol) and 4M hydrochloric acid-ethyl acetate solution (10 mL) and stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, diethyl ether was added to the residue, and the precipitate was filtered and dried. The resulting crude product was dissolved in methanol (10 mL), passed through a syringe filter (pore size 0.45 μm), concentrated under reduced pressure, and dried to obtain a white solid (yield 1.27 g, yield 96%). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.90 - 3.04 (m, 2 H), 3.18 (dd, J=13.82, 7.70 Hz, 1 H), 3.31 - 3.37 (m, 1 H), 3.59 (s, 3 H), 3.72 (s, 3 H), 4.19 (t, J=6.72 Hz, 1 H), 4.43 - 4.51 (m, 1 H), 6.82 - 6.88 (m, 2 H), 7.16 - 7.23 (m, 2 H), 7.63 (m, J=8.80 Hz, 2 H), 8.12 - 8.24 (m, 2 H), 8.45 (br s, 3 H), 9.38 (d, J=7.34 Hz, 1 H); MS (ESI-TOF) m / z: calcd for C 20 H 24 N3O6[M + H] + 402.1660; found 402.1654.

[0076] Test Example 1. Stability test against human carboxyesterase 1 The stability of the compounds synthesized above against human carboxyesterase 1 was evaluated. Specifically, the procedure was as follows.

[0077] A 300 μM compound solution was prepared as needed by diluting a DMSO stock solution (30 mM) of the compound with Tris-HCl buffer (pH 7.4). 50 μL of Tris-HCl buffer containing 0.8 μg / mL human carboxyesterase 1 (4920-CE, R&D Systems Inc., Minneapolis, USA) and 50 μL of the 300 μM compound solution were mixed and incubated at 37 °C. After 24 hours, 5 μL of 1 M hydrochloric acid was added to stop the reaction, and 30 μL was injected into an HPLC to determine the peak area of ​​the compound at 220 nm. Human carboxyesterase 1, which had been previously mixed with hydrochloric acid, was mixed with the compound solution and analyzed by HPLC. The peak area at 0 hours was used as the peak area, and the ratio of this to the area at 24 hours was calculated using Equation 1 (Remaining amount (%) = (Compound peak area at 24 hours) / (Compound peak area at 0 hours) × 100) to determine the remaining amount.

[0078] The results are shown in Figure 1. The compounds in the examples were found to be stable against esterase.

[0079] Test Example 2. Evaluation of antimicrobial activity against Porphyromonas gingivalis. The antibacterial activity of the compounds synthesized above against Porphyromonas gingivalis was evaluated. Specifically, the following procedure was followed.

[0080] To a bacterial suspension of Porphyromonas gingivalis W83, each compound was added after being dissolved in DMSO and diluted twice in series to a final concentration of 0.025–200 μM. A control solution of 0.2% DMSO without the compounds was added. For comparison, azithromycin (AZM) and chlorhexidine (CHX) were prepared similarly and added to the cell suspension. The bacterial concentration was 10 6 The cells were cultured at CFU / mL in modified GAM medium under anaerobic conditions at 37°C for 72 hours. The minimum inhibitory concentration (MIC) was measured by the microdilution method. The minimum bactericidal concentration (MBC) was measured after seeding the cell suspension onto blood agar and culturing it under anaerobic conditions at 37°C for 48 hours.

[0081] The results are shown in Table 1. The units of the values ​​in Table 1 are μM. The compounds in the examples showed strong antibacterial activity against Porphyromonas gingivalis.

[0082] [Table 1]

[0083] Test Example 3. Inhibitory effect on the growth of oral bacteria. The inhibitory effect of the compounds synthesized above on the growth of oral bacteria was evaluated. Specifically, the following procedure was followed.

[0084] 10 6Various oral bacteria with CFU / mL concentrations were cultured under the following conditions, and their MICs (minimum inhibitory concentration) were measured by microdilution. Each compound was dissolved in DMSO and added to cell suspensions using a serial 2-fold dilution method to achieve final concentrations of 0.025–200 μM. 0.2% DMSO without the compound was added as a control. Azithromycin (AZM) was used as a comparative agent, and was prepared similarly and added to cell suspensions. Porphyromonas gingivalis W83: Incubated in modified GAM medium under anaerobic conditions at 37°C for 72 hours. Fusobacterium nucleatum ATCC 25586: Incubated in modified GAM medium under anaerobic conditions at 37°C for 72 hours. Prevotella intermedia ATCC 25611: Incubated in modified GAM medium under anaerobic conditions at 37°C for 48 hours. Aggregatibacter actinomycetencomitans JP2: Incubated in TSB medium supplemented with 6 mg / mL yeast extract and 0.4 mg / mL sodium bicarbonate under anaerobic conditions at 37°C for 24 hours. Streptococcus mitis ATCC 903: Cultured in BHI medium under aerobic conditions at 37°C for 24 hours.

[0085] The results are shown in Table 2. The units of the values ​​in Table 2 are μM. Compound 1 in the example showed a weak growth inhibitory effect on P. intermedia in addition to Porphyromonas gingivalis. Compounds 2 and 3 did not show a growth inhibitory effect on oral bacteria other than Porphyromonas gingivalis, and showed Porphyromonas gingivalis specificity.

[0086] [Table 2]

[0087] Test Example 4. Cytotoxicity Test on Oral Epithelial Cell Lines The cytotoxicity of the synthesized compound described above was evaluated against an oral-derived epithelial cell line. Specifically, it was carried out as follows.

[0088] The human oral-derived epithelial cell line Ca9-22 was seeded in a 96-well plate at 10 5 cells / well, and each compound prepared by dissolving in DMSO to a final concentration of 50 - 200 μM was added. As a control, 0.2% DMSO without the compound was added, and the cell viability was set at 100%. Also, as a comparison target, azithromycin (AZM) was used. The Ca9-22 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 50 U / mL penicillin, and 50 μg / mL streptomycin at 37 °C under 5% CO2 for 24 hours. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] Assay was performed to measure OD570, and the cell viability (Survival rate%) was evaluated from Equation 2 (Cell viability (%) = "OD570 (containing the compound)" / "OD570 (without the compound)" × 100).

[0089] The results are shown in Figure 2. The compounds of the examples did not show cytotoxicity against animal cells.

Claims

1. General formula (1): 【Chemistry 1】 [In the formula: R 1 R represents a halogen atom or a nitro group. 2 This indicates an alkoxy group or an acylamino group. A compound represented by the same, or a salt thereof, or a solvate thereof.

2. R 2 The compound according to claim 1, or a salt thereof, or a solvate thereof, wherein is an alkoxy group having 1 to 4 carbon atoms or an acylamino group having 2 to 5 carbon atoms.

3. The compound according to claim 1, or a salt thereof, or a solvate thereof, wherein the halogen atom is a chlorine atom.

4. R 1 is a halogen atom and R 2 is an alkoxy group, or R 1 is a nitro group and R 2 The compound according to claim 1, or a salt thereof, or a solvate thereof, wherein is an alkoxy group or an acylamino group.

5. R 1 is a chlorine atom and R 2 is an alkoxy group having 1 to 4 carbon atoms, or R 1 is a nitro group and R 2 is an alkoxy group having 1 to 4 carbon atoms or an acylamino group having 2 to 5 carbon atoms, the compound or a salt thereof or a solvate thereof according to claim 1.

6. An antimicrobial agent containing a compound or salt thereof, or a solvate thereof, according to any one of claims 1 to 5.

7. The antimicrobial agent according to claim 6, wherein the target bacterium is Porphyromonas gingivalis.

8. An agent for preventing or improving bacterial infections, comprising a compound or salt thereof, or a solvate thereof, as described in any one of claims 1 to 5.

9. The preventive or corrective agent according to claim 8, wherein the bacterial infection is periodontal disease.

10. An oral composition containing a compound or salt thereof, or a solvate thereof, according to any one of claims 1 to 5.

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