Synthesis method of naphthyridine-4-ketone-3-carboxylic acid compound and application thereof in antibacterial drugs

Abstract

This invention belongs to the field of novel compound synthesis and antibacterial drug technology, and discloses a method for synthesizing naphthidine-4-one-3-carboxylic acid compounds and their application in antibacterial drugs. The general structural formula is as follows: wherein the naphthidine ring is a 1,8-naphthidine ring or a benzo[ b Naphthidine ring; R 1 It is a halogen or alkoxy group; R 2 It is cycloalkyl or aralkyl; the compound includes its pharmaceutically acceptable salt, hydrate, solvate, or prodrug. The benzo[…] obtained in this invention… b Naphthidone-3-carboxylic acid derivatives have excellent antibacterial activity and have the potential to be developed into drugs for the treatment of Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Shigella flexneri.

Description

Technical Field

[0001] This invention belongs to the field of novel compound synthesis and antibacterial drug technology, and in particular to a method for synthesizing naphthidine-4-one-3-carboxylic acid compounds and their application in antibacterial drugs. Background Technology

[0002] The 1,8-naphthidine-4-one skeleton, as an important structural unit in nitrogen-containing heterocyclic compounds, has attracted much attention in the field of medicinal chemistry due to its broad biological activity. This skeleton belongs to the azabicyclic system, and its unique electron distribution and spatial configuration enable it to specifically interact with a variety of biological targets. This invention demonstrates that 1,8-naphthidine-4-one compounds exhibit potential application value in multiple therapeutic areas, including antitumor, antihistamine, anti-inflammatory, and antibacterial applications, reflecting the multifunctionality of this skeleton as a dominant structural unit. The 1,8-naphthidine-4-one-3-carboxylic acid compound core contains a pyridine ring; replacing the pyridine ring with a quinoline ring yields benzo[…]. b Naphthoidone compounds. In 2001, Michel Tabart first synthesized a series of benzo[ b A novel topical antibacterial agent of the naphthidone class was developed, and its antibacterial activity was evaluated. This invention is the first report of this type of structure, and the resulting compound exhibits antibacterial activity against Gram-positive bacteria. However, the synthetic route is lengthy and cumbersome, involving a total of 10 reaction steps, and only yields a 7-position derivative, posing a significant challenge to the rapid construction of compound libraries and subsequent process development.

[0003] In summary, this invention establishes a new, efficient, and flexible synthetic method, overcoming the limitations of existing preparation routes. This invention, through two optimized synthetic routes, synthesizes eight novel benzo[...] genus... b The preparation of naphthidinone-3-carboxylic acid derivatives provides a comprehensive methodological reference for the efficient synthesis and structural modification of such skeletal derivatives. Furthermore, the antibacterial activity of a series of 1,8-naphthidin-4-one derivatives was assessed based on this method.

[0004] In addition, some synthetic methods for 1,8-naphthidine-4-one compounds have been disclosed in related technologies, but no systematic inventions have been made on their antibacterial activity, nor has the application potential of these compounds in the field of antibacterial has been clarified, and their antibacterial value has not yet been explored. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for synthesizing naphthidine-4-one-3-carboxylic acid compounds and their application in antibacterial drugs.

[0006] The technical solution adopted by this invention to solve its technical problem is: A naphthidin-4-one-3-carboxylic acid compound has the following general structural formula: Wherein, the naphthidine ring is a 1,8-naphthidine ring or a benzo[ b Naphthidine ring; R 1 It is a halogen or alkoxy group; R 2 It is a cycloalkyl or aralkyl group; the compound includes its pharmaceutically acceptable salt, hydrate, solvate or prodrug.

[0007] Furthermore, its structural formula is one of the following: .

[0008] The method for synthesizing naphthidine-4-one-3-carboxylic acid compounds as described above uses β-keto ester 8 as a key intermediate, which is oxidized to obtain nitrogen oxide 9. Nitrogen oxide 9 undergoes an amination reaction with an amine to generate compound 11. Compound 11 is cyclized to generate compound 12. Compound 12 is detert-butylated under acidic conditions to obtain the target naphthidine-4-one-3-carboxylic acid derivative 13.

[0009] Furthermore, the synthetic route of the method is as follows: .

[0010] Furthermore, the β-keto ester 8 is prepared by any of the following routes: Pathway 1: Starting with quinoline, the product undergoes C3-H bond acylation, aromatization, hydrolysis, and condensation to generate Weinreb amide 5, which is then mediated by the condensation of Claisen esters. Pathway 2: Starting with 2-nitrobenzaldehyde, it is reduced and cyclized with ethyl 3,3-diethoxypropionate, and then mediated to undergo Claisen ester condensation to generate the product. The three key steps in the above preparation process—nitrogen oxidation, 2-position amination, and cyclization—correspond to the following reagents: The nitrogen oxidation reaction uses m-chloroperoxybenzoic acid m CPBA or urea peroxide UHP, 2-position amination reaction using tripyrroleylphosphonium hexafluorophosphate bromide or trifluoromethanesulfonic anhydride Tf₂O, cyclization reaction using N , N -Dimethylformamide dimethyl acetal DMF-DMA.

[0011] Further, the specific steps of the first path are as follows: starting with quinoline, acylation of C3-H bond is used to generate compound 2, aromatization is performed under the action of DDQ to obtain compound 3, hydrolysis with sodium hydroxide is used to generate compound 4, condensation with EDCI is used to generate Weinreb amide 5, and LDA is used to mediate the condensation of tert-butyl acetate with Claisen ester to generate β-keto ester 8. The reaction pathway is as follows: Among them, R 1 For hydrogen and halogen, R 2 It can be cycloalkyl or aralkyl.

[0012] Further, the specific steps of the second path are as follows: using 2-nitrobenzaldehyde as the starting material, it is reduced by stannous chloride and cyclized with ethyl 3,3-diethoxypropionate to generate compound 7, and LDA-mediated condensation with tert-butyl acetate to generate β-keto ester 8. The reaction pathway is as follows: Among them, R 1 For halogens or alkoxy groups, R 2 It is a cycloalkyl group.

[0013] The application of naphthidine-4-one-3-carboxylic acid compounds as described above in the preparation of antibacterial drugs.

[0014] Furthermore, the drug is an antibacterial drug used to treat infections caused by Gram-positive and / or Gram-negative pathogens.

[0015] Furthermore, the Gram-positive pathogens include one or more selected from Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), and the Gram-negative pathogens include one or more selected from Escherichia coli and Shigella flexneri.

[0016] The advantages and positive effects of this invention are as follows: 1. The benzo[] obtained in this invention b Naphthidone-3-carboxylic acid derivatives have excellent antibacterial activity and have the potential to be developed into drugs for the treatment of Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Shigella flexneri.

[0017] 2. This invention provides a route A using inexpensive and readily available quinoline derivatives as starting materials, with good substrate versatility. Furthermore, through screening of the oxidant system, a raw material recycling reaction strategy, and a novel Tf₂O / DBU-mediated amination system, the reaction yield is significantly improved, overcoming the bottlenecks of low reactivity and high reagent consumption in traditional amination reactions.

[0018] 3. The two routes in this invention complement each other: Route A has low raw material costs and good substrate universality, and is suitable for the large-scale preparation of most derivatives; Route B has short steps and simple operation, and can realize diversified modification of the skeleton at multiple sites. Together, they fill the gap in the invention of general synthetic methods and system material basis for this type of skeleton.

[0019] 4. Among the derivatives prepared by the method of this invention, compound 13kb has a MIC of 0.16–0.31 μg / mL against Staphylococcus aureus and 1.25 μg / mL against enoxacin, thus 13kb exhibits 4–8 times higher activity than enoxacin; furthermore, the inhibition zone diameter of 13kb is 35.0 mm, superior to enoxacin's 29.5 mm. Compound 13kb has a MIC of 0.18 μg / mL against Shigella flexneri and 0.31 μg / mL against enoxacin, thus 13kb exhibits approximately 2 times higher activity than enoxacin. This provides a convenient, efficient, practical, and feasible synthetic method and high-quality candidate molecules for the development of novel antibacterial drugs.

[0020] 5. Among the derivatives prepared by the method of the present invention, compound 13kb has significantly better antibacterial activity against drug-resistant bacteria than existing drugs: against methicillin-resistant Staphylococcus aureus (MRSA): the MIC of compound 13kb is 0.08 μg / mL, while that of enoxacin is 0.63 μg / mL. Therefore, the activity of 13kb is about 8 times higher than that of enoxacin.

[0021] 6. Among the derivatives prepared by the method of the present invention, compound 13kb exhibits low cytotoxicity. Attached Figure Description

[0022] Figure 1 This is the 1H NMR spectrum of compound 13gd in deuterated chloroform + trifluoroacetic acid in this invention; Figure 2 This is the carbon NMR spectrum of compound 13gd in deuterated dimethyl sulfoxide in this invention; Figure 3 This is the 1H NMR spectrum of compound 13hb in deuterated trichloromethane in this invention; Figure 4 This is the carbon NMR spectrum of compound 13hb in deuterated trichloromethane in this invention; Figure 5 This is the 1H NMR spectrum of compound 13ib in deuterated chloroform in this invention; Figure 6 This is the carbon NMR spectrum of compound 13ib in deuterated chloroform in this invention; Figure 7 This is the 1H NMR spectrum of compound 13jb in deuterated trichloromethane in this invention; Figure 8This is the carbon NMR spectrum of compound 13jb in deuterated trichloromethane in this invention; Figure 9 This is the 1H NMR spectrum of compound 13kb in deuterated chloroform in this invention; Figure 10 The image shows the carbon NMR spectrum of compound 13kb in deuterated chloroform. Figure 11 The image shows the 1H NMR spectrum of compound 13lb in deuterated chloroform. Figure 12 The image shows the carbon NMR spectrum of compound 13lb in deuterated chloroform. Figure 13 This is the 1H NMR spectrum of compound 13mb in deuterated dimethyl sulfoxide in this invention; Figure 14 This is the carbon NMR spectrum of compound 13mb in deuterated dimethyl sulfoxide (DMSO). Figure 15 This is the 1H NMR spectrum of compound 13nb in deuterated trichloromethane in this invention; Figure 16 This is the carbon NMR spectrum of compound 13nb in deuterated trichloromethane in this invention; Figure 17 These are the toxicity test results of compounds 13eb, 13kb, and 13lb in this invention on LX2 human hepatic stellate cells. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0024] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0025] A naphthidin-4-one-3-carboxylic acid compound has the following general structural formula: Wherein, the naphthidine ring is a 1,8-naphthidine ring or a benzo[ b Naphthidine ring; R 1 It is a halogen or alkoxy group; R 2 It is a cycloalkyl or aralkyl group; the compound includes its pharmaceutically acceptable salt, hydrate, solvate or prodrug.

[0026] Furthermore, its structural formula is one of the following: .

[0027] The method for synthesizing naphthidine-4-one-3-carboxylic acid compounds as described above uses β-keto ester 8 as a key intermediate, which is oxidized to obtain nitrogen oxide 9. Nitrogen oxide 9 undergoes an amination reaction with an amine to generate compound 11. Compound 11 is cyclized to generate compound 12. Compound 12 is detert-butylated under acidic conditions to obtain the target naphthidine-4-one-3-carboxylic acid derivative 13.

[0028] Furthermore, the synthetic route of the method is as follows: .

[0029] Furthermore, the β-keto ester 8 is prepared by any of the following routes: Pathway 1: Starting with quinoline, the product undergoes C3-H bond acylation, aromatization, hydrolysis, and condensation to generate Weinreb amide 5, which is then mediated by the condensation of Claisen esters. Pathway 2: Starting with 2-nitrobenzaldehyde, it is reduced and cyclized with ethyl 3,3-diethoxypropionate, and then mediated to undergo Claisen ester condensation to generate the product. The three key steps in the above preparation process—nitrogen oxidation, 2-position amination, and cyclization—correspond to the following reagents: The nitrogen oxidation reaction uses m-chloroperoxybenzoic acid ( m CPBA or urea peroxide (UHP), the 2-position amination reaction uses tripyrrolidinyl phosphonium hexafluorophosphate bromide or trifluoromethanesulfonic anhydride (Tf2O), and the ring-closure reaction uses... N , N -Dimethylformamide dimethyl acetal (DMF-DMA).

[0030] Further, the specific steps of the first path are as follows: starting with quinoline, acylation of C3-H bond is used to generate compound 2, aromatization is performed under the action of DDQ to obtain compound 3, hydrolysis with sodium hydroxide is used to generate compound 4, condensation with EDCI is used to generate Weinreb amide 5, and LDA is used to mediate the condensation of tert-butyl acetate with Claisen ester to generate β-keto ester 8. The reaction pathway is as follows: Among them, R 1 For hydrogen and halogen, R 2 It can be cycloalkyl or aralkyl.

[0031] Further, the specific steps of the second path are as follows: using 2-nitrobenzaldehyde as the starting material, it is reduced by stannous chloride and cyclized with ethyl 3,3-diethoxypropionate to generate compound 7, and LDA-mediated condensation with tert-butyl acetate to generate β-keto ester 8. The reaction pathway is as follows: Among them, R 1 For halogens or alkoxy groups, R 2 It is a cycloalkyl group.

[0032] The application of naphthidine-4-one-3-carboxylic acid compounds as described above in the preparation of antibacterial drugs.

[0033] Furthermore, the drug is an antibacterial drug used to treat infections caused by Gram-positive and / or Gram-negative pathogens.

[0034] Furthermore, the Gram-positive pathogens include one or more selected from Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), and the Gram-negative pathogens include one or more selected from Escherichia coli and Shigella flexneri.

[0035] Route A: Starting with quinoline, compound 2 is generated through dearomatization. Subsequently, compound 2 is re-aromatized with DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) to give compound 3. Compound 3 is hydrolyzed in sodium hydroxide solution to convert to compound 4. Compound 4 then undergoes condensation with EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) to generate Weinreb amide 5. This amide 5 then reacts with tert-butyl acetate in the presence of lithium diisopropylamino(LDA) to generate β-keto ester 8. β-keto ester 8 is then reacted with m-chloroperoxybenzoic acid (…). m Compounds 9 and 10 are oxidized with CPBA or urea peroxide (UHP) to generate nitrogen oxides 9; subsequently, compounds 9 and 10 are amination with tripyrrolidinyl phosphonium hexafluorophosphate (PyBroP) or trifluoromethanesulfonic anhydride (Tf₂O) to generate 11. Then... N,N 12 is cyclized with dimethylformamide dimethyl acetal (DMF-DMA), and finally the tert-butyl group is removed under acidic conditions to generate compound 13.

[0036] Route B uses 2-nitrobenzaldehyde as the starting material, reacting it with stannous chloride dihydrate and ethyl 3,3-diethoxypropionate to generate compound 7. Subsequently, it undergoes a Claisen ester condensation reaction with tert-butyl acetate in the presence of lithium diisopropylaminolithium (LDA) to generate β-keto ester 8. β-keto ester 8 is then reacted with m-chloroperoxybenzoic acid (… mCompounds 9 and 10 are oxidized with CPBA or urea peroxide (UHP) to generate nitrogen oxides 9; subsequently, compounds 9 and 10 are amination with tripyrrolidinyl phosphonium hexafluorophosphate (PyBroP) or trifluoromethanesulfonic anhydride (Tf₂O) to generate 11. Then... N,N 12 is cyclized with dimethylformamide dimethyl acetal (DMF-DMA), and finally the tert-butyl group is removed under acidic conditions to generate compound 13.

[0037] Using the above method, the structures and numbering of the compounds synthesized in this invention are as follows: Meanwhile, to expand the application scope of these known compounds, explore their potential medicinal value, and fill gaps in existing technologies, this invention supplements the systematic antibacterial activity determination of some 1,8-naphthidine-4-one-3-carboxylic acid compounds, clarifying key information such as the strength and range of their antibacterial activity. This invention not only enriches the application scenarios of these parent compounds but also complements the antibacterial invention of novel compounds, jointly providing important experimental data support for the development and application of 1,8-naphthidine-4-one-3-carboxylic acid compounds in the field of antibacterial drugs.

[0038] .

[0039] Specifically, the relevant preparation and testing methods are as follows: Common operation 1: Under argon protection, the substituent (R) will be... 1 It is hydrogen, 6-halogen, R 2 A solution of tetrahydrofuran (12.22 mL) of quinoline derivative 1 (6.11 mmol) (containing cycloalkyl and aralkyl groups) was added to a dry double-necked flask and cooled to 0 °C. A boron trifluoride diethyl ether complex (6.72 mmol) was added dropwise, and the mixture was stirred for 15 min. The temperature was then lowered to -50 °C, and a solution of tert-butylmagnesium chloride (1 M in tetrahydrofuran, 7.3 mL, 7.33 mmol) was added dropwise. The mixture was stirred for 30 min, and then trifluoroacetic anhydride (12.22 mmol) and trifluoroacetic acid (18.33 mmol) were added dropwise at -50 °C. The mixture was then transferred to room temperature and stirred. After the reaction was complete, the reaction was quenched with saturated potassium carbonate solution, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the target crude product 2.

[0040] Common operation 2: An ethanol solution of the crude product of compound 2 (6.11 mmol) was added to a dry flask, followed by the addition of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (12.22 mmol). Concentrated hydrochloric acid (12 M, 2 mL, 18.33 mmol) was added dropwise, and the mixture was heated to 80 °C. After the reaction was complete, the reaction was quenched with a saturated sodium carbonate solution, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the crude product. The target product 3 was purified by column chromatography (petroleum ether:ethyl acetate = 20:1~5:1, v / v) to obtain the crude product.

[0041] Common operation 3: Compound 3 (5.16 mmol) and an aqueous solution of sodium hydroxide (4 M, 3.4 mL, 13.42 mmol) were added to a dried 25 mL flask and stirred at 120 °C for 6 h. The reaction mixture was then cooled to room temperature. The pH of the reaction mixture was adjusted to 3 by adding 3 M hydrochloric acid solution. The mixture was filtered to obtain a solid, which was washed with water (3 × 5 mL). The filtrate was extracted with n-butanol (3 × 15 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a yellowish-brown solid product 4.

[0042] Common operation 4: Add to a solution of compound 4 (17.3 mmol) in dichloromethane (70 mL) N,O -Dimethylhydroxylamine hydrochloride (25.95 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (25.95 mmol), and 4-dimethylaminopyridine (25.95 mmol). The mixture was stirred at room temperature for 6 hours until the reaction was complete. The reaction solution was diluted with saturated sodium chloride solution and extracted three times with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under vacuum to give the crude product. Purification by column chromatography (petroleum ether:ethyl acetate = 10:1 to 1:1, v / v) gave the target product 5.

[0043] Common operation 5: With substituents (R) 1An ethanol solution (83 mL) of o-nitrobenzaldehyde 6 (16.56 mmol, 5-halogen, 7-halogen, or alkoxy) was prepared by adding stannous chloride dihydrate (66.24 mmol) and ethyl 3,3-diethoxypropionate (41.4 mmol) sequentially. The resulting solution was then stirred at 90 °C for 4 h. After the reaction was complete, the reaction solution was concentrated under vacuum, quenched with saturated sodium bicarbonate solution, filtered through diatomaceous earth, washed with EA, and the filtrate was washed three times with saturated sodium chloride solution. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 20:1–5:1, v / v) to obtain the target product.

[0044] Common operation 6: A solution of diisopropylamine (42.39 mmol) in anhydrous tetrahydrofuran (85 mL) was added to a dry flask purged with argon and equipped with a magnetic stir bar and a rubber diaphragm stopper, and the mixture was cooled to -78 °C. A solution of n-butyllithium in n-hexane (1.6 M, 27 mL, 42.39 mmol) was added dropwise, and the mixture was stirred at the same temperature for 30 minutes. Then, tert-butyl acetate (42.39 mmol) was added dropwise, and the mixture was stirred at the same temperature for 1 hour. A solution of compound 7 (15.7 mmol) in anhydrous tetrahydrofuran (65 mL) was added to the above reaction mixture, and the mixture was stirred at -78 °C for 30 minutes. The mixture was then transferred to a -20 °C bath and stirred until TLC showed complete reaction. The reaction was quenched with saturated ammonium chloride aqueous solution and extracted three times with ethyl acetate. The organic phases were combined, washed successively with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the crude product. The crude product was obtained by column chromatography (petroleum ether:ethyl acetate = 100:1 ~ 10:1, v / v) to give the target product 8.

[0045] Common operation 7: A solution of diisopropylamine (42.39 mmol) in anhydrous tetrahydrofuran (85 mL) was added to a dry flask purged with argon and equipped with a magnetic stir bar and a rubber diaphragm stopper, and the mixture was cooled to -78°C. A solution of n-butyllithium in n-hexane (1.6 M, 27 mL, 42.39 mmol) was added dropwise, and the mixture was stirred at the same temperature for 30 minutes. Then, tert-butyl acetate (42.39 mmol) was added dropwise, and the mixture was stirred at the same temperature for 1 hour. A solution of compound 5 (15.7 mmol) in anhydrous tetrahydrofuran (65 mL) was added to the above reaction mixture, and the mixture was stirred continuously at -78°C for 10 minutes, at which point TLC showed that the reaction was complete. The reaction was quenched with saturated ammonium chloride aqueous solution and extracted three times with ethyl acetate. The organic phases were combined, washed successively with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 100:1 to 10:1, v / v) to obtain the target product 8.

[0046] General operation 8: A solution of compound 8 (12.3 mmol) in dichloromethane (41 mL) was placed in an ice-water bath, and then m-chloroperoxybenzoic acid (14.76 mmol) was slowly added. The resulting mixture was then stirred overnight at room temperature. The solution was concentrated under vacuum and then subjected to column chromatography (petroleum ether:ethyl acetate = 10:1 ~ pure ethyl acetate, v / v) to give target product 9.

[0047] Common operation 9: Phthalic anhydride (8.40 mmol) and urea peroxide (9.10 mmol) were added sequentially to a dichloromethane (24 mL) solution of compound 8 (5.88 mmol), and the resulting mixture was stirred overnight at room temperature. The mixture was concentrated under vacuum and then subjected to column chromatography (petroleum ether:ethyl acetate = 10:1 ~ pure ethyl acetate, v / v) to give target product 9.

[0048] Common operations 10: In a dry, double-necked flask equipped with a magnetic stir bar and sealing film, add 4 Å molecular sieve (same mass as compound 9), compound 9 (1.4 mmol), and dichloroethane (5.6 mL). Then add the substituted (R... 2An amine compound 10 (3.5 mmol) of cyclopropyl or benzyl group, triethylamine (4.2 mmol), and tripyrrolidinyl phosphonium hexafluorophosphate bromide (2.66 mmol) were added. The resulting mixture was stirred at room temperature for several hours until TLC showed complete reaction. The reaction mixture was filtered, the mother liquor was diluted with saturated aqueous NH4Cl solution, and extracted three times with dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated under vacuum to give a crude product. The target product 11 was purified by column chromatography (petroleum ether:ethyl acetate = 100:1 to 10:1, v / v) to obtain the target product 11.

[0049] Common operations 11: In a dry flask filled with argon gas, equipped with a magnetic stir bar and a rubber stopper, the compound was added sequentially with ethyl acetate (5.4 mL) solution 9 (2.67 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (5.34 mmol). The mixture was stirred at -0°C, and trifluoromethanesulfonic anhydride (4.00 mmol) was added dropwise, followed by stirring at room temperature for 6 h. The reaction was quenched with saturated sodium bicarbonate aqueous solution and extracted three times with dichloroethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the crude product. Purification by column chromatography (petroleum ether:ethyl acetate = 100:1 to 10:1, v / v) yielded the target product 11.

[0050] Common operations 12: In a sealed tube equipped with a magnetic stir bar, add a 1 mL solution of toluene containing 0.4 mmol of compound 11. Add... N,N - Dimethyl methyl ester dimethyl acetal (N,N-dimethylformamide dimethyl acetal, 0.72 mmol), and then the resulting solution was stirred at 100 °C for 3 h. After cooling to room temperature, it was concentrated under vacuum to obtain the crude product. The target product 12 was purified by column chromatography (petroleum ether:ethyl acetate = 10:1~EA, v / v) to obtain the target product 12.

[0051] Common operations 13: Trifluoroacetic acid (0.52 mL) was added to a solution of compound 12 (0.26 mmol) in dichloromethane (0.52 mL), and the resulting solution was stirred at room temperature for 1 h. The solution was concentrated under vacuum and azeotropically treated with ethanol to remove residual trifluoroacetic acid. The crude product was washed with n-pentane and filtered to obtain the target product.

[0052] The following examples illustrate this in detail. Example 1: 1-Benzyl-4-oxo-1,4-dihydrobenzo[ b Synthesis of [1,8]naphthyl-3-carboxylic acid The synthesis method of Example 1 is as follows: The synthesis started with quinoline-3-carboxylic acid, which underwent a condensation reaction via general operation 4 to generate compound 5g; subsequently, compound 5g reacted via general operation 7 to give compound 8g; compound 8g underwent an oxidation reaction via general operation 3 to generate compound 9g; based on this, compound 9g and benzylamine 10d were reacted via general operation 10 to generate compound 11gd; then, compound 12gd was generated via general operation 12; finally, tert-butyl was removed under acidic conditions via general operation 13 to generate compound 13gd.

[0053] Final yield: 100%, yellow solid. Figure 1 , Figure 2 As shown, thin-layer chromatography: R f = 0.21 (dichloromethane:MeOH = 20:1). Melting point: 222-228℃. Structural parameters: 1 H NMR (600 MHz, CDCl3+ CF3COOH) δ 10.00(s, 9H), 9.49 (s, 1H), 9.31 (s, 1H), 8.24 (d, J = 9.6 Hz, 1H), 8.17 (d, J = 8.3Hz, 1H), 8.03 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.37 (q, J = 8.1, 7.3 Hz, 3H), 5.96 (s, 2H). 13 C NMR (151 MHz, DMSO- d 6 ) δ177.30, 166.94, 152.03, 151.25, 150.98, 139.66, 136.19, 130.19,129.58, 129.17, 128.72, 127.53, 127.48, 125.42, 124.35, 123.79, 118.72,113.16, 53.77. HRMS (+ESI-TOF) m / z [M+H] + Calcd for C 20 H 15 N2O3331.1077; found331.1072. Example 2: 7-Chloro-1-cyclopropyl-4-oxo-1,4-dihydrobenzo[ b Synthesis of [1,8]naphthyl-3-carboxylic acid The synthesis method of Example 2 is as follows: The synthesis started with 6-chloroquinoline, which underwent a dearomatization reaction via general operation 1 to generate compound 2h. Subsequently, compound 2h was rearomatized via general operation 2 in the presence of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to give compound 3h. Compound 3h was hydrolyzed via general operation 3 in sodium hydroxide solution to convert it into compound 4h. Based on this, compound 4h underwent condensation via general operation 4 in the presence of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) to generate Weinreb amide 5h. Then, via general operation 7, it reacted with tert-butyl acetate in the presence of lithium diisopropylamino(LDA) to generate β-keto ester 8h. β-Ketoester 8h was oxidized by m-chloroperoxybenzoic acid via general operation 8 to generate nitrogen oxides 9h; next, compound 9h and cyclopropylamine 10b were amination with tripyrroleylphosphonium hexafluorophosphate (PyBroP) via general operation 10 to generate 11hb. Then, via general operation 12... N,N 12hb is generated by cyclization of dimethylformamide dimethyl acetal (DMF-DMA), and finally, the tert-butyl group is removed under acidic conditions through general operation 13 to generate compound 13hb.

[0054] Final yield: 86%, yellow solid. Figure 3 , Figure 4 As shown, thin-layer chromatography: R f = 0.19 (EA). Melting point: 254-259℃. Structural parameters: 1 H NMR (600 MHz, CDCl3) δ 14.07 (s, 1H), 9.35 (s, 1H), 9.09 (s, 1H), 8.23 ​​(dd, J = 9.3, 5.1 Hz, 1H), 7.79 – 7.70 (m, 2H), 3.97 (tt, J = 7.6,4.2 Hz, 1H), 1.45 – 1.40 (m, 2H), 1.20 – 1.15 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 177.27, 165.93, 150.26, 150.02, 149.81, 131.54, 129.86, 127.95, 127.15,125.52, 125.14, 119.72, 108.74, 37.90, 9.02. HRMS (+ESI-TOF) m / z [M+H] + Calcdfor C 16 H 12 ClN2O3315.0530; found 315.0530. Example 3: 7-Bromo-1-cyclopropyl-4-oxo-1,4-dihydrobenzo[ b Synthesis of [1,8]naphthyl-3-carboxylic acid The synthesis method of Example 3 is as follows: The synthesis started with 6-bromoquinoline, which underwent a dearomatization reaction via general operation 1 to generate compound 2i. Compound 2i was then re-aromatized via general operation 2 in the presence of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to give compound 3i. Compound 3i was hydrolyzed via general operation 3 in sodium hydroxide solution to convert to compound 4i. Compound 4i then underwent a condensation reaction via general operation 4 in the presence of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) to generate Weinreb amide 5i. This amide then reacted with tert-butyl acetate via general operation 7 in the presence of lithium diisopropylamino(LDA) to generate β-keto ester 8i. β-keto ester 8i was oxidized by urea peroxide via general operation 9 to generate nitric oxide 9i. Next, compound 9i reacted with cyclopropylamine 10b via general operation 11 in the presence of trifluoromethanesulfonic anhydride (Tf₂O) to generate 11ib. Finally, it was subjected to general operation 12... N,N12ib is cyclized from dimethylformamide dimethyl acetal (DMF-DMA) and then detert-butylated under acidic conditions via general operation 13 to generate compound 13ib.

[0055] Final step yield: 99%, yellow solid. Figure 5 and Figure 6 As shown, thin-layer chromatography: R f = 0.53 (PE:EA = 1:1). Melting point: 260-269℃. Structural parameters: 1 H NMR (600 MHz, CDCl3) δ 14.01 (s, 1H), 9.29 (s,1H), 9.08 (s, 1H), 8.15 (d, J = 9.1 Hz, 1H), 8.09 (d, J = 2.3 Hz, 1H), 7.88 (dd, J = 9.1, 2.3 Hz, 1H), 3.95 (tt, J = 7.5, 4.2 Hz, 1H), 1.41 (t, J = 7.0 Hz, 2H),1.19 – 1.14 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ HRMS (+ESI-TOF) m / z [M+H] + Calcd for C 16 H 12 BrN2O3360.1812; found360.1812. Example 4: 8-Bromo-1-cyclopropyl-4-oxo-1,4-dihydrobenzo[ b Synthesis of [1,8]naphthyl-3-carboxylic acid The synthesis method of Example 4 is as follows: The synthesis started with 4-bromo-2-nitrobenzaldehyde. Following general procedure 1, the compound 7j was generated by reacting stannous chloride dihydrate and ethyl 3,3-diethoxypropionate. Subsequently, following general procedure 2, it underwent a Claisen ester condensation reaction with tert-butyl acetate in the presence of lithium diisopropylamino (LDA) to generate a β-keto ester 8j. β-keto ester 8j was oxidized by urea peroxide in general procedure 9 to generate nitrogen oxides 9j. Next, compound 9j reacted with cyclopropylamine 10b in general procedure 11, undergoing amination in the presence of trifluoromethanesulfonic anhydride (Tf₂O) to generate 11jb. Finally, following general procedure 12... N,N - Dimethylformamide dimethyl acetal (DMF-DMA) is cyclized to generate 12jb, and finally, under acidic conditions, the tert-butyl group is removed through general operation 13 to generate compound 13jb.

[0056] Final step yield: 99%, yellow solid. Figure 7 and Figure 8 As shown, thin-layer chromatography: R f = 0.53 (PE:EA = 1:1). Melting point: 298-306℃. Structural parameters: 1 H NMR (600 MHz, CDCl3) δ 14.07 (s, 1H), 9.35 (s,1H), 9.09 (s, 1H), 8.09 (d, J = 9.0 Hz, 1H), 7.76 (d, J = 2.5 Hz, 1H), 7.60 (dd, J = 9.0, 2.5 Hz, 1H), 3.97 (tt, J = 7.6, 4.2 Hz, 1H), 1.45-1.40 (m, 2H), 1.20-1.15 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 177.31, 165.93, 151.02, 149.95, 146.81,129.27, 129.01, 127.24, 124.43, 123.96, 123.39, 118.27, 108.74, 37.90, 9.02.HRMS (+ESI-TOF) m / z [M+H] + Calcd for C 15 H 12 BrN2O3359.0025; found 359.0025. Example 5: 1-Cyclopropyl-7-fluoro-4-oxo-1,4-dihydrobenzo[ b Synthesis of [1,8]naphthyl-3-carboxylic acid The synthesis method of Example 5 is as follows: The synthesis started with 6-fluoroquinoline, which underwent a dearomatization reaction via general operation 1 to generate compound 2k. Compound 2k was then re-aromatized via general operation 2 in the presence of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to give compound 3k. Compound 3k was hydrolyzed via general operation 3 in sodium hydroxide solution to convert to compound 4k. Compound 4k then underwent a condensation reaction via general operation 4 in the presence of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) to generate Weinreb amide 5k. This amide then reacted with tert-butyl acetate via general operation 7 in the presence of lithium diisopropylamino(LDA) to generate β-keto ester 8k. β-keto ester 8k was oxidized by urea peroxide via general operation 9 to generate nitric oxide 9k. Next, compound 9k reacted with cyclopropylamine 10b via general operation 11 in the presence of trifluoromethanesulfonic anhydride (Tf₂O) to generate 11kb. Finally, it was subjected to general operation 12... N,N - Dimethylformamide dimethyl acetal (DMF-DMA) is cyclized to generate 12kb, and finally, under acidic conditions, the tert-butyl group is removed through general operation 13 to generate compound 13kb.

[0057] Final step yield: 72%, yellow solid. Figure 9 and Figure 10 As shown, thin-layer chromatography: R f = 0.22 (EA). Melting point: 247-251℃. Structural parameters: 1 H NMR (600 MHz, CDCl3) δ 14.07 (s, 1H), 9.35 (s, 1H), 9.09 (s, 1H), 8.23 ​​(dd, J = 9.3, 5.1 Hz, 1H), 7.79 – 7.70 (m, 2H), 3.97 (tt, J =7.6, 4.2 Hz, 1H), 1.45 – 1.40 (m, 2H), 1.20 – 1.15 (m, 2H). 13 C NMR (150 MHz, CDCl3) δ178.73, 165.23, 160.40, 158.73, 149.92, 147.33, 145.82, 136.78,136.74, 130.70, 130.63, 125.65, 125.58, 123.80, 123.63, 119.10, 110.72,110.58, 106.32, 34.00, 6.77. HRMS (+ESI-TOF) m / z [M+H] + Calcd forC 16 H 12 FN2O3299.0826; found 299.0828. Example 6: 1-Cyclopropyl-6-fluoro-4-oxo-1,4-dihydrobenzo[ b Synthesis of [1,8]naphthyl-3-carboxylic acid The synthesis method of Example 6 is as follows: The synthesis started with 2-fluoro-6-nitrobenzaldehyde. Following general procedure 1, compound 7l was generated by reacting stannous chloride dihydrate and ethyl 3,3-diethoxypropionate. Subsequently, following general procedure 2, tert-butyl acetate underwent a Claisen ester condensation reaction to generate β-keto ester 8l. β-keto ester 8l was then reacted with m-chloroperoxybenzoic acid (...) m CPBA) is oxidized to produce nitrogen oxide 9l; next, compound 9l and cyclopropylamine 10b undergo amination via general operation 11 in the presence of trifluoromethanesulfonic anhydride (Tf₂O) to produce 11lb. Then, via general operation 12... N,N 12 lb of dimethylformamide dimethyl acetal (DMF-DMA) was cyclized and then the tert-butyl group was removed under acidic conditions via general operation 13 to generate compound 13 lb.

[0058] Final step yield: 72%, yellow solid. Figure 11 and Figure 12 As shown, thin-layer chromatography: R f = 0.22 (EA). Melting point: 239-245℃. Structural parameters: 1 H NMR (600 MHz, CDCl3) δ 14.07 (s, 1H), 9.35 (s, 1H), 9.09 (s, 1H), 7.55 (dd, J= 9.0, 8.5 Hz, 1H), 7.48 (ddd, J = 8.5, 8.5, 5.0 Hz,1H), 7.32 (d, J = 8.5 Hz, 1H), 3.97 (tt, J = 7.6, 4.2 Hz, 1H), 1.45-1.40 (m, 2H), 1.20-1.15 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 177.22, 165.93, 161.02, 159.03,152.05, 151.24, 151.17, 148.98, 128.39, 128.32, 126.75, 126.69, 122.73,118.08, 116.04, 114.40, 114.30, 112.83, 112.66, 109.20, 37.90, 9.02. HRMS (+ESI-TOF) m / z [M+H] + Calcd for C 16 H 12 FN2O3299.0826; found 299.0819. Example 7: 1-Cyclopropyl-7-methoxy-4-oxo-1,4-dihydrobenzo[ b Synthesis of [1,8]naphthyl-3-carboxylic acid The synthesis method of Example 7 is as follows: The synthesis started with 5-methoxy-2-nitrobenzaldehyde. Following general procedure 1, stannous chloride dihydrate and ethyl 3,3-diethoxypropionate reacted to generate compound 7m. Subsequently, following general procedure 2, tert-butyl acetate underwent a Claisen ester condensation reaction to generate β-keto ester 8m. β-keto ester 8m was then reacted with m-chloroperoxybenzoic acid (...) m CPBA) is oxidized to produce nitrogen oxide 9m; next, compound 9m reacts with cyclopropylamine 10b via general operation 11 in the presence of trifluoromethanesulfonic anhydride (Tf₂O) to undergo amination to produce 11mb. Then, via general operation 12... N,N 12mb was cyclized with dimethylformamide dimethyl acetal (DMF-DMA) to generate compound 12mb, and then the tert-butyl group was removed under acidic conditions via general operation 13 to generate compound 13mb.

[0059] Final step yield: 99%, yellow solid. Figure 13 and Figure 14 As shown, thin-layer chromatography: R f = 0.53 (PE:EA = 1:1). Melting point: 256-259℃. Structural parameters: 1 H NMR (600 MHz, CDCl3) δ 14.01 (s, 1H), 9.29 (s,1H), 9.08 (s, 1H), 8.15 (d, J = 9.1 Hz, 1H), 8.09 (d, J = 2.3 Hz, 1H), 7.88 (dd, J = 9.1, 2.3 Hz, 1H), 3.95 (tt, J = 7.5, 4.2 Hz, 1H), 1.41 (t, J = 7.0 Hz, 2H),1.19 – 1.14 (m, 2H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 177.20, 165.90, 155.71, 150.39,149.82, 149.69, 131.41, 127.30, 123.86, 120.41, 119.00, 108.97, 108.00,56.34, 37.87, 9.02.. HRMS (+ESI-TOF) m / z [M+Na] + Calcd for C 17 H 15 N2O4Na333.0845; found 333.0848. Example 8: 1-Cyclopropyl-8-methoxy-4-oxo-1,4-dihydrobenzo[ b Synthesis of [1,8]naphthyl-3-carboxylic acid The synthesis method of Example 8 is as follows: The synthesis started with 4-methoxy-2-nitrobenzaldehyde. Following general procedure 1, it reacted with stannous chloride dihydrate and ethyl 3,3-diethoxypropionate to generate compound 7n. Subsequently, following general procedure 2, it underwent a Claisen ester condensation reaction with tert-butyl acetate in the presence of lithium diisopropylamino (LDA) to generate β-keto ester 8n. β-keto ester 8n was then reacted with m-chloroperoxybenzoic acid (...) m CPBA) is oxidized to generate nitrogen oxide 9n; next, compound 9n reacts with cyclopropylamine 10b via general operation 11 in the presence of trifluoromethanesulfonic anhydride (Tf₂O) to undergo amination to generate 11nb. Then, via general operation 12... N,N - Dimethylformamide dimethyl acetal (DMF-DMA) is cyclized to generate 12nb, and finally, under acidic conditions, the tert-butyl group is removed through general operation 13 to generate compound 13nb.

[0060] Final step yield: 99%, yellow solid. Melting point: 244-247℃. Figure 15 and Figure 16 As shown, the structural parameters are: 1 HNMR (600 MHz, CDCl3) δ 14.01 (s, 1H), 9.29 (s, 1H), 9.08 (s, 1H), 7.30 (d, J =8.5 Hz, 1H), 7.23 (dd, J = 8.5, 8.5 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 3.96 (s,3H), 3.95 (tt, J = 7.5, 4.2 Hz, 1H), 1.41 (t, J = 7.0 Hz, 2H), 1.19 – 1.14 (m,2H). 13 C NMR (151 MHz, CDCl3) δ HRMS (+ESI-TOF) m / z [M+Na] + Calcd for C 17 H 15 N2O4Na 333.0845; found 333.0850. In vitro antibacterial activity test of the compound: Experimental strains: The test strains selected included common clinical pathogens and drug-resistant strains, specifically Staphylococcus aureus (Staphylococcus aureus). S. aureus ), Escherichia coli ( E. coli ), methicillin-resistant Staphylococcus aureus (MRSA) and Shigella flexneri ( Shigella flexneri Among them, Staphylococcus aureus is a common Gram-positive pathogen, Escherichia coli is a common Gram-negative pathogen, MRSA is a methicillin-resistant Staphylococcus aureus strain, and Shigella flexneri is the main pathogen causing bacterial dysentery.

[0061] inhibition zone determination: This experiment was conducted strictly in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines for antimicrobial susceptibility testing. The entire procedure was performed in a sterile biosafety cabinet, and all consumables and culture media were autoclaved at 121°C and 0.1 MPa for 30 min. Standard strains of Staphylococcus aureus and Escherichia coli, stored at low temperatures, were activated by streaking on LB agar. Single colonies were picked and inoculated onto LB broth, incubated at 37°C with shaking until the logarithmic growth phase, and the OD600 of the bacterial suspension was adjusted to 0.05 with fresh LB agar for later use.

[0062] Accurately weigh the target compound and dissolve it in dimethyl sulfoxide to prepare a 1 mg / mL test solution. After sterilization through a 0.22 μm sterile filter membrane, accurately pipette 10 μL of the solution and evenly add it to a 6 mm diameter sterile blank filter paper disc. Let it stand at room temperature in a clean bench until the solvent has completely evaporated. Prepare blank control filter paper discs and positive control filter paper discs containing 10 μL of sterile dimethyl sulfoxide using the same method. Spread 100 μL of bacterial suspension evenly on LB solid agar plates and let it stand for 5 min until the bacterial suspension is completely absorbed. Then, flatten the filter paper discs on the surface of the plate, with a spacing of ≥24 mm between each filter paper disc and a distance of ≥15 mm from the edge of the plate. Incubate upside down at 37°C for 16–18 h. Measure the diameter of the inhibition zone vertically and crosswise with vernier calipers. Set up 3 replicates for each group, and express the results as the average value.

[0063] Minimum inhibitory concentration (MIC): Dissolve 1.8 g of nutrient broth (NB) powder in 100 mL of ultrapure water and autoclave at 121 °C to obtain sterile nutrient broth medium. Incubate the frozen bacterial culture at -20 °C for 24 h, then at 2–4 °C for 10 h. After the culture temperature returns to room temperature, add the bacterial solution to 50 mL of NB medium and incubate at 37 °C. Prepare a 10 μmol / mL solution of the test compound using dimethyl sulfoxide (DMSO). Dilute the high-concentration test compound solution to different concentrations using LB agar and place the solutions in 96-well plates. Dilute the bacterial solution (OD600 ≈ 0.05) 1000 times to obtain a concentration of approximately 10. 5 The bacterial suspensions were mixed with compound solutions of different concentrations and then incubated at 37°C for 18 h. The MIC value of the compound was determined by observing the clarity of the solution.

[0064] Table 1. 1,8-Naphthid-4-one-3-carboxylic acid and benzo[ b Assay for the antibacterial activity of naphthidone-3-carboxylic acid derivatives

[0065] Note: In the table, the larger the value of the inhibition zone diameter, the stronger the antibacterial activity; the smaller the value of the minimum inhibitory concentration, the stronger the antibacterial activity; "NA" indicates that no antibacterial activity was detected, "-" indicates that this item was not detected, and ">" indicates that the antibacterial effect was not achieved at this concentration; Enoxacin is a positive control drug; The 1,8-naphthidine-4-one-3-carboxylic acid derivatives involved in this invention can effectively inhibit the growth of Gram-positive and Gram-negative pathogenic bacteria and have excellent antibacterial activity. Among them, compounds 13eb, 13gb, 13kb, 13lb, and 13nb all show good antibacterial effects against Staphylococcus aureus, Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and Shigella flexneri. Some compounds have better inhibitory activity against drug-resistant bacteria than the positive control drug enoxacin and can be used in antibacterial drugs for the treatment of bacterial infections.

[0066] Compound cytotoxicity testing: Experimental cells: The experiment used LX2 human hepatic stellate cells as a normal human cell model to evaluate the in vitro biosafety of the target derivative. The main reagents included MTT reagent and dimethyl sulfoxide (DMSO), and the positive control drug was enoxacin, a commonly used clinical antibiotic.

[0067] Thiazol blue assay for cell proliferation inhibition: To systematically evaluate the in vitro biosafety of the compounds of this invention, the thiazolyl blue assay was used to determine the inhibitory effect of the target compounds obtained from the previous antibacterial activity screening on the proliferation of human hepatic stellate cells at the set test concentrations. Cell viability in each group was calculated, and the results are as follows: Figure 17 As shown.

[0068] LX2 human hepatic stellate cells in the logarithmic growth phase were collected and a cell suspension was prepared in Durbeco Modified Eagle (DMEM) high-glucose liquid medium containing 10% fetal bovine serum and 100 units / mL penicillin-streptomycin mixture. The cell concentration was adjusted to 6 × 10⁻⁶ cells / mL. 5 After achieving a cell count / mL, the cells were seeded into 96-well culture plates at a seeding volume of 100 μL per well, resulting in a final cell count of 6 × 10⁶ cells per well. 4 Cells were cultured at 37°C in a 5% CO2 incubator for 12-16 h until complete cell adhesion. Target compound solutions were added to a final concentration of 10 μmol / L (consistent with the effective concentration used in the previous antibacterial activity screening), with 100 μL of the compound working solution (prepared using the above-mentioned serial dilution of complete culture medium) added to each well. A blank control group was also set up: LX2 cells were seeded only, and the same volume of DMEM high-glucose complete culture medium containing 0.1% dimethyl sulfoxide (DMS) was added as the drug-treated group, with no added compound, at a concentration of 100 μL / well; and an enoxacin positive control group was established: enoxacin was diluted to a final concentration of 10 μmol / L using the above-mentioned complete culture medium, with 100 μL added to each well. Dimethyl sulfoxide (DMSO) for all compound dosing systems: First, a high-concentration stock solution of 10 mmol / L was prepared by dissolving the target compound and enoxacin reference standard. Before administration, it was serially diluted with complete culture medium to a working concentration of 10 μmol / L, ensuring that the final volumetric concentration of DMSO in all dosing systems was strictly controlled within 0.1% (far below the cytotoxicity threshold). After culturing for 24 h, MTT solution was added to each well: at a concentration of 5 mg / mL, 10 μL of MTT solution was added to each well at a ratio of 10 μL of MTT solution per 100 μL of culture medium. After incubation for 4 h, the supernatant in the wells was discarded, and 150 μL of analytical grade DMSO was added to each well, followed by shaking on a low-speed shaker for 10 min. Complete dissolution of formazan crystals: Formazan crystals are blue-purple insoluble products formed by the reaction of succinate dehydrogenase in the mitochondria of living cells with MTT reagent during incubation at 37°C for 4 h, and they deposit inside the cells. The dissolution process must ensure complete dissolution of the crystals, with no visible particles remaining; otherwise, it will significantly affect the accuracy of the absorbance detection results. The absorbance value of each well at a wavelength of 490 nm was measured using an ELISA reader. The cell viability rate of each group was calculated according to the formula: Cell viability (%) = (Absorbance value of the drug treatment group / Absorbance value of the blank control group) × 100%.

[0069] Experimental Results and Analysis: like Figure 17 As shown, the results of the thiazolyl blue cell proliferation inhibition experiment indicate that the benzo[…] of the present invention… b The naphthidone-3-carboxylic acid compounds 13kb, 13eb, and 13lb did not exhibit significant inhibitory effects on the proliferation of human hepatic stellate cells at the set test concentrations, demonstrating good in vitro biocompatibility. Specifically, compounds 13kb and 13eb showed cell viability rates of 100.6% and 95.3%, respectively, with no significant difference from the blank control group, and their cell viability rates were significantly higher than the positive control drug enoxacin (90.0%). Compound 13lb showed a cell viability rate of 88.5%, also exhibiting extremely low cytotoxicity.

[0070] In this experiment, the final concentration of dimethyl sulfoxide in all compound administration systems was strictly controlled below 2%, and the solvent had little effect on cell proliferation, making the experimental results reliable.

[0071] The compounds 13kb, 13eb, and 13lb of this invention possess both excellent antibacterial activity and good in vitro biosafety, with a wide safety window. They can be used as novel antibacterial compounds to prepare antibacterial drugs, or as lead compounds for subsequent in vivo pharmacodynamic and toxicological studies.

[0072] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A naphthidin-4-one-3-carboxylic acid compound, characterized in that: Its general structural formula is as follows: Wherein, the naphthidine ring is a 1,8-naphthidine ring or a benzo[ b Naphthidine ring; R 1 It is a halogen or alkoxy group; R 2 It is a cycloalkyl or aralkyl group; the compound includes its pharmaceutically acceptable salt, hydrate, solvate or prodrug.

2. The naphthidin-4-one-3-carboxylic acid compound according to claim 1, characterized in that: Its structural formula is one of the following: 。 3. The method for synthesizing naphthidin-4-one-3-carboxylic acid compounds as described in claim 1 or 2, characterized in that: The method uses β-keto ester 8 as a key intermediate, which is oxidized to obtain nitrogen oxide 9. Nitrogen oxide 9 undergoes an amination reaction with an amine to generate compound 11. Compound 11 is cyclized to generate compound 12. Compound 12 is detert-butylated under acidic conditions to obtain the target naphthidin-4-one-3-carboxylic acid derivative 13.

4. The synthesis method according to claim 3, characterized in that: The synthetic route of the method is as follows: 。 5. The synthesis method according to claim 3, characterized in that: The β-keto ester 8 is prepared by any of the following routes: Pathway 1: Starting with quinoline, the product undergoes C3-H bond acylation, aromatization, hydrolysis, and condensation to generate Weinreb amide 5, which is then mediated by the condensation of Claisen esters. Pathway 2: Starting with 2-nitrobenzaldehyde, it is reduced and cyclized with ethyl 3,3-diethoxypropionate, and then mediated to undergo Claisen ester condensation to generate the product. The three key steps in the above preparation process—nitrogen oxidation, 2-position amination, and cyclization—correspond to the following reagents: The nitrogen oxidation reaction uses m-chloroperoxybenzoic acid m CPBA or urea peroxide UHP, 2-position amination reaction using tripyrroleylphosphonium hexafluorophosphate bromide or trifluoromethanesulfonic anhydride Tf₂O, cyclization reaction using N , N -Dimethylformamide dimethyl acetal DMF-DMA.

6. The synthesis method according to claim 5, characterized in that: The specific steps of the first path are as follows: starting with quinoline, acylation of C3-H bond generates compound 2,2,3-dichloro-5,6-dicyanobenzoquinone, aromatization under the action of DDQ to obtain compound 3, hydrolysis with sodium hydroxide to obtain compound 4, condensation of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with EDCI to generate Weinreb amide 5, and condensation of lithium diisopropylaminoethyl with tert-butyl acetate with Claisen ester mediated by LDA to generate β-keto ester 8. The reaction pathway is as follows: Among them, R 1 For hydrogen and halogen, R 2 It can be cycloalkyl or aralkyl.

7. The synthesis method according to claim 5, characterized in that: The specific steps of the second path are as follows: using 2-nitrobenzaldehyde as the starting material, it is reduced by stannous chloride and cyclized with ethyl 3,3-diethoxypropionate to generate compound 7, and LDA-mediated condensation with tert-butyl acetate to generate β-keto ester 8. The reaction pathway is as follows: Among them, R 1 For halogens or alkoxy groups, R 2 It is a cycloalkyl group.

8. The use of the naphthidin-4-one-3-carboxylic acid compounds as described in claim 1 or 2 in the preparation of antibacterial drugs.

9. The application according to claim 8, characterized in that: The drug is an antibacterial agent used to treat infections caused by Gram-positive and / or Gram-negative pathogens.

10. The application according to claim 9, characterized in that: The Gram-positive pathogens include one or more selected from Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), and the Gram-negative pathogens include one or more selected from Escherichia coli and Shigella flexneri.

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