CN115894522A

CN115894522A - Synthesis method and application of triazole penicillin-containing molecules

Info

Publication number: CN115894522A
Application number: CN202211734323.4A
Authority: CN
Inventors: 王雪松; 李友宾; 崔雪; 徐俊裕; 王燕; 马健婷; 唐瑶
Original assignee: Hainan Medical College
Current assignee: Hainan Medical College
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-04-04

Abstract

The invention discloses a synthesis method and application of triazole penicillin molecules, which are used for synthesizing a compound CX-1 or/and a compound CX-2 or/and a compound CX-3 after protecting carboxyl on 6-aminopenicillanic acid.

Description

Synthesis method and application of triazole penicillin-containing molecules

Technical Field

The invention relates to the field of triazole penicillin-containing molecules, in particular to a synthesis method and application of triazole penicillin-containing molecules.

Background

Since penicillin has been found to be useful in the treatment of bacterial infections, more and more penicillins have been used clinically, and resistance has developed in bacteria over time, the synthesis of new penicillins or new analogues with antibacterial activity has been slow. In the previous literature research, a triazole skeleton is found to exist in antibacterial drugs, for example, triazole is introduced to the methyl of the 2-position of sulbactam, so that the beta-lactamase inhibitor, namely tazobactam, which is widely applied so far is obtained, and the triazole skeleton is more stable and has better anti-infection effect than sulbactam. Therefore, the triazole and the 6-APA are connected by an activity splicing principle to synthesize a penicillin molecule containing the triazole, and the capability of inhibiting the activity of the beta-lactamase and the antibacterial capability of the penicillin molecule are measured.

Disclosure of Invention

Therefore, the invention provides a synthesis method of triazole penicillin-containing molecules, which solves the problems.

(1) Protection of the carboxyl group on 6-aminopenicillanic acid

6-aminopenicillanic acid (1g, 4.6 mmol) and triethylamine (0.929292g, 9.2mmol) were added to a 50mL eggplant-shaped flask containing anhydrous dichloromethane (10 mL) and stirred until dissolved, and ethyl acetoacetate (0.598 g,4.6 mmol) was further added and stirring was continued for 3 hours. The solvent was then removed in vacuo, 9-bromofluorene (1.127g, 4.6 mmol) was added dissolved in DMF (10 mL) and the reaction mixture was stirred overnight, after which the solution was diluted with ethyl acetate and washed with brine (3 × 15 mL), the combined organic layers were dried over anhydrous magnesium sulfate, concentrated under reduced pressure and separated by silica gel column chromatography (eluent: PE/EtOAc =20/1,15/1,10/1, v/v) to give a white solid (compound 2). Compound 2 was dissolved in acetone (10 mL), and after addition of p-toluenesulfonic acid monohydrate (0.969 g, 5.1mmol) for 1h a white precipitate appeared, which was filtered and the filter cake washed with diethyl ether (5 mL). Finally, the filter cake was dissolved in dichloromethane (20 mL), triethylamine (0.9292g, 9.2mmol) was added and stirred for 2h, further NaOH solution (1m, 10ml) was added to separate the organic phase, the aqueous phase was extracted with dichloromethane, all the organic phases were combined, washed with brine (10 mL), dried and concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =3/1,2/1, v/v) to give a white powder (compound 3, 0.35g, 20%).

(2) Synthesis of Compound CX-1

Bis (triphenylphosphine) palladium dichloride (0.21g, 0.3mmol), benzoyl chloride (1.4g, 10mmol), phenylacetylene (1.276g, 11mmol) and triethylamine (3.5 mL) are added into a 100mL eggplant-shaped bottle filled with 30mL dichloromethane, and finally cuprous iodide (0.19g, 1mmol) is added and reacted for 12 hours under the protection of argon. The reaction mixture was filtered through celite, extracted 3 times with water (3 × 40 mL), the organic layer was dried, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =500/1,400/1,300/1,v/v) to give a tan solid (compound 4). Compound 4 (1.03g, 5 mmol) and ethyl p-aminobenzoate (0.825g, 5 mmol) were sequentially added to a 50mL eggplant-shaped bottle, followed by addition of 10mL of methanol and refluxing at 70 ℃ for 6 hours. The solvent was dried by spinning and separated by silica gel column chromatography (eluent: PE/EtOAc =30/1,25/1,20/1, v/v) to give a yellow powder (compound 5,1.06g, 57%). After compound 5 (0.371g, 1mmol) and lithium tert-butoxide (0.24g, 3mmol) were sequentially added to a 25mL eggplant-shaped bottle, 4mL of dichloromethane and p-toluenesulfonyl azide (TsN 3,0.394g, 2mmol) were added and reacted at room temperature for 12h and the TLC detection reaction was substantially completed, extraction was performed 3 times with water (3X 20 mL) and dichloromethane (20 mL), dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =20/1,15/1,10/1, v/v) to obtain a white solid (compound 6,0.23g, yield 78%). Compound 6 (0.1465g, 0.5mmol) was dissolved in 4mL of methanol, followed by dropwise addition of a sodium hydroxide solution (0.2g, 5mmol, dissolved in 0.5mL of water), reaction at room temperature for 12h, completion of the reaction by TLC, adjustment of the pH to about 2 with a 2NHCl solution, extraction with water (20 mL) and ethyl acetate (3X 20 mL) for 3 times, drying over anhydrous magnesium sulfate, and concentration under reduced pressure to obtain a white solid (Compound 7,0.136g, 97% yield). Compound 7 (0.1325g, 0.5mmol), HATU (0.22g, 0.58mmol) were dissolved in 2mL dmf, DIPEA (0.075g, 0.58mmol) was added dropwise, after reaction at room temperature for 30min, compound 3 (0.152g, 0.4mmol) was added, the reaction was overnight, after the reaction was almost completed by TLC detection, extraction was performed 3 times with water (3 × 20 mL) and ethyl acetate (20 mL), drying was performed with anhydrous magnesium sulfate, concentration was performed under reduced pressure, and separation was performed by silica gel column chromatography (eluent: PE/EtOAc =3/1,2/1, v/v) to obtain a yellow oily liquid (compound 8,0.23g, yield 92%). Compound 8 (0.3135g, 0.5mmol), naHCO3 (0.084g, 1mmol) were added to a mixed solution of THF (2 mL) and water (2 mL), finally Pd/C (1.0 g) was added, a hydrogen balloon was attached to the reaction flask, reaction was carried out at room temperature for 12h, pd/C was filtered and washed with a mixed solution of THF (1 mL) and water (1 mL), citric acid (0.126g, 0.6 mmol) was added to acidify for 30min, water (5 mL) and ethyl acetate (3 × 5 mL) were added to extract 3 times, the organic layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: ethyl acetate/petroleum ether =2/1,3/1,1 ch3cooh, v/v) to give a pale yellow oily liquid (compound CX-1,0.095g, 40% yield).

(3) Synthesis of Compound CX-2

Bis (triphenylphosphine) palladium dichloride (0.21g, 0.3mmol), benzoyl chloride (1.4g, 10mmol), phenylacetylene (1.276g, 11mmol) and triethylamine (3.5 mL) are added into a 100mL eggplant-shaped bottle filled with 25mL dichloromethane, and finally cuprous iodide (0.19g, 1mmol) is added and reacted for 12 hours under the protection of argon. The reaction mixture was filtered through celite, extracted 3 times with water (3 × 40 mL), the organic layer was dried, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =500/1,400/1,300/1,v/v) to give a tan solid (compound 4). Compound 4 (1.03g, 5 mmol) and ethyl p-aminobenzoate (0.825g, 5 mmol) were sequentially added to a 50mL eggplant-shaped bottle, followed by addition of 10mL of methanol and refluxing at 70 ℃ for 6 hours. The solvent was dried by spinning and separated by silica gel column chromatography (eluent: PE/EtOAc =30/1,25/1,20/1, v/v) to give a yellow powder (compound 5,1.06g, 57%). Compound 5 (0.742g, 2mmol) was dissolved in 5mL acetonitrile, and p-toluenesulfonylazide (TsN 3,0.788g, 4mmol) and DBU (0.608g, 4mmol) were added dropwise thereto, reacted at room temperature for 2h, and after checking that the reaction was substantially complete by TLC, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =20/1,15/1,10/1, v/v) to give a white powder (Compound 9,0.508g, 64% yield). Compound 9 (0.393972 g, 1mmol), sodium borohydride (0.074g, 2mmol) were dissolved in 4mL of ethanol, reacted at room temperature for 4h, after completion of the reaction was checked by TLC, quenched with water, extracted 3 times with ethyl acetate (3X 10 mL), and the organic layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =5/1,4/1,3/1, v/v) to give a white powder (Compound 10,0.356g, 89% yield). Compound 10 (0.2g, 0.5 mmol), indole (0.0877g, 0.75mmol) were dissolved in 2mL of LPCM, and after anhydrous ferric trichloride (0.04g, 0.25mmol) was added, the reaction was refluxed at 60 ℃ for 12h, and checked by TLC for substantial completion, the reaction was separated by silica gel column chromatography (eluent: PE/EtOAc =10/1,8/1,6/1, v/v) to obtain a white powder (Compound 11,0.229g, yield 92%). Compound 11 (0.4 g, 0.8mmol) was dissolved in 4mL of methanol, followed by dropwise addition of a sodium hydroxide solution (0.328 g,8mmol in 0.5mL of water), reaction at room temperature for 12h, completion of the reaction by TLC, adjustment of the pH to about 2 with a 2N Cl solution, extraction with water (20 mL) and ethyl acetate (3X 20 mL) for 3 times, drying of the organic layer over anhydrous magnesium sulfate, and concentration under reduced pressure to give a white solid (Compound 12,0.38g, 97% yield). Compound 12 (0.235g, 0.5 mmol), HATU (0.22g, 0.58mmol) were dissolved in 2mL dmf, DIPEA (0.075g, 0.58mmol) was added dropwise, after reaction at room temperature for 30min, compound 3 (0.152g, 0.4 mmol) was added, the reaction was overnight, after the TLC detection reaction was substantially complete, extracted 3 times with water (3 × 20 mL) and ethyl acetate (20 mL), the organic layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =3/1,2/1, v/v) to give a yellow oily liquid (compound 13,0.316g, yield 95%). Compound 13 (0.25g, 0.3mmol), naHCO3 (0.05g, 0.6 mmol) were added to a mixed solution of THF (1.5 mL) and water (1.5 mL), and finally Pd/C (0.6 g) was added, a hydrogen balloon was attached to the reaction flask, the reaction was carried out at room temperature for 12h, pd/C was filtered and washed with a mixed solution of THF (1 mL) and water (1 mL), after adding citric acid (0.084g, 0.4mmol) and acidifying for 30min, water (5 mL) and ethyl acetate (3X 5 mL) were added and extracted 3 times, the organic layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: ethyl acetate/petroleum ether =2/1,3/1,1 CH3COOH, v/v) to give a pale yellow liquid (Compound CX-2,0.101g, 50% yield).

(4) Synthesis of Compound CX-3

Bis-triphenylphosphine palladium dichloride (0.21g, 0.3mmol), benzoyl chloride (1.4g, 10mmol), phenylacetylene (1.276g, 111mmol) and triethylamine (3.5 mL) were sequentially added into a 100mL eggplant-shaped bottle filled with 25mL dichloromethane, and finally cuprous iodide (0.19g, 1mmol) was added and reacted for 12 hours under the protection of argon. The reaction solution was filtered through celite, extracted 3 times with water (3 × 40 mL), dried, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =500/1,400/1,300/1,v/v) to give a tan solid (compound 4). Compound 4 (1.03g, 5 mmol) and ethyl p-aminobenzoate (0.825g, 5 mmol) were sequentially added to a 50mL eggplant-shaped bottle, followed by addition of 10mL of methanol and refluxing at 70 ℃ for 6 hours. The solvent was dried by spinning off and separated by silica gel column chromatography (eluent: PE/EtOAc =30/1,25/1,20/1, v/v) to give a yellow powder (compound 5,1.06g, 57%). Compound 5 (0.742g, 2mmol) was dissolved in 5mL acetonitrile, p-toluenesulfonylazide (TsN 3,0.788g, 4mmol) and DBU (0.608g, 4mmol) were added dropwise, reacted at room temperature for 2h, detected by TLC, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =20/1,15/1,10/1, v/v) to give a white powder (compound 9,0.508g, yield 64%). Compound 9 (0.3970 g, 1mmol), sodium borohydride (0.074g, 2mmol) were dissolved in 4mL ethanol, reacted at room temperature for 4h, detected by TLC, quenched with water, extracted 3 times with ethyl acetate (3X 10 mL), dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =5/1,4/1,3/1, v/v) to give a white powder (compound 10,0.356g, 89% yield). Compound 10 (0.12g, 0.3mmol), 1,3, 5-trimethoxybenzene (0.0756g, 0.45mmol) were dissolved in 2ml of LPCM, and finally anhydrous ferric chloride (0.024g, 0.15mmol) was added, and after refluxing at 60 ℃ for 12h, detection by TLC, separation by silica gel column chromatography (eluent: PE/EtOAc =10/1,8/1,6/1, v/v) was carried out to obtain a white powder (compound 14,0.138g, yield 84%). Compound 14 (0.1647g, 0.3mmol) was dissolved in 3mL of methanol, and sodium hydroxide solution (0.12g, 3mmol, 0.5mL of water) was added dropwise thereto, reacted at room temperature for 12h, after TLC detection, adjusted to pH 2 with 2N HCl solution, extracted 3 times with water (20 mL) and ethyl acetate (3X 20 mL), dried, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE EtOAc =3/1,2/1,1 CH3COOH, v/v) to give a white solid (Compound 15,0.1g, 64% yield). Compound 15 (0.24pg, 0.47mmol), HATU (0.205g, 0.54mmol) were dissolved in 2mL of DMF, DIPEA (0.07g, 0.54mmol) was added dropwise thereto, and after reaction at room temperature for 30min, compound 3 (0.14g, 0.37mmol) was added thereto, the reaction was allowed to proceed overnight, after TLC detection, extraction was performed 3 times with water (3X 20 mL) and ethyl acetate (20 mL), dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =3/1,2/1, v/v) to obtain a yellow oily liquid (Compound 16,0.196g, yield 60%). Compound 16 (0.106g, 0.12mmol), naHCO3 (0.02g, 0.24mmol) were added to a mixed solution of THF (1.5 mL) and water (1.5 mL), and finally 5% Pd/C (0.24 g) was added, a hydrogen balloon was attached to the reaction flask, the reaction was carried out at room temperature for 12h, the Pd/C was filtered and washed with a mixed solution of THF (1 mL) and water (1 mL), citric acid (0.042g, 0.2mmol) was added to acidify for 30min, then water (5 mL) and ethyl acetate (3 × 5 mL) were added to extract 3 times, the organic layer was dried, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: ethyl acetate/petroleum ether =2/1,3/1, 1. CH3COOH, v/v), to give a pale yellow liquid (compound CX-3,0.045g, yield 52%).

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, triazole and 6-APA are connected by an active splicing principle to synthesize penicillin molecules containing triazole. The penicillin molecule containing triazole prepared by the invention has obvious inhibition effect on beta-lactamase, wherein CX-1 has inhibition effect on beta-lactamase when the concentration of a compound CX-1 is 4 mu M, 0.1 mu M or 0.01 mu M, the optimum concentration is 0.1 mu M, and the inhibition rate is 15.53%. The compound CX-20.1 μ M is used as the concentration, and the inhibition rate is 7.16%.

Drawings

FIG. 1-synthetic route for protection of the carboxyl group on APA

FIG. 2 synthetic route of compound CX-1

FIG. 3 Synthesis route of Compound CX-2

FIG. 4 Synthesis route of Compound CX-3

FIG. 5 Mass Spectroscopy of Compound CX-1

FIG. 6 Mass Spectroscopy of Compound CX-2

FIG. 7 Mass Spectroscopy of Compound CX-3

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

Experimental reagent

6-aminopenicillanic acid (6-APA), annaiji chemical; ethyl acetoacetate, annagi chemical; 9-bromofluorene, schenssi Biochemical technology Ltd, tianjin; dichloromethane, chemical industry of west longu; n, N-dimethylformamide, annaigig chemistry; para-toluenesulfonic acid monohydrate, annaiji chemical; acetone, guangzhou chemical reagent plant; triethylamine, chemical industry of west longe; ether, guangzhou chemical laboratories; sodium hydroxide, GALAXY-REAGENT; petroleum ether, GALAXY-REAGENT; ethyl acetate, GALAXY-REAGENT; anhydrous magnesium sulfate, chemical industry of west longu; bis (triphenylphosphine) palladium dichloride, benzoyl chloride, adamas-beta; phenylacetylene, adamas-beta; cuprous iodide, annagi chemical; ethyl p-aminobenzoate, adamas-beta company; methanol, chemical industry of west longu; lithium tert-butoxide, annagi chemical; p-toluenesulfonyl azide, annaghi chemical; hydrochloric acid; 2- (7-Azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), adamas-beta; n, N-Diisopropylethylamine (DIPEA), alatin; sodium bicarbonate, chemical industry of west longu; tetrahydrofuran, chemical industry of west longridge; pd/C, annaiji chemistry; citric acid, mao chemical reagent factory, tianjin; acetonitrile, alatin corporation; 1, 8-diazabicycloundec-7-ene (DBU), adamas-beta; sodium borohydride, chemical industry of julongsan; ethanol, chemical industry of juju de; indole, annagi chemistry; anhydrous ferric chloride, annaiji chemical; 1,3,5-trimethoxybenzene, adamas-beta.

Laboratory apparatus

Circulating water vacuum pump (SHZ-DIII type), step City Zeng Yuanha instruments, LLC; a diaphragm vacuum pump (model MVP-1000), shanghai Iran instruments, inc.; a low-temperature cooling liquid circulating pump (DLSB-5 type), which consolidates the municipal Provisional Instrument, limited liability company; rotary evaporator (model N-1300), shanghai elan instruments ltd; ultrasonic cleaning apparatus (KQ-100V type), ultrasonic instruments ltd, kunshan city; magnetic heating stirrer (model 78-1), australian instruments, inc. of Changzhou; analytical balance (XS 105 DR), mettler TOLEDO; electronic balance (FA 1104 type), shanghai liangping instruments liability ltd; an air drying oven (DHG-9240A type), shanghai seiki instruments ltd; dark box type ultraviolet analyzer (model ZF-20D), sweden instruments & Equipment Ltd of Steheny; LCD digital control heating type magnetic stirrer (MS-H-Pro), bluespin; thin layer chromatography silica gel plate (HSGF 254), cigarette platform jiangyou silica gel development ltd; melting point apparatus (WRR type), shanghai apparatus electro-physical optics instruments ltd; nuclear magnetic resonance spectrometer (AVANCE 600), bruker corporation.

Example 1

(1) Protection of the carboxyl group on 6-APA

6-APA (1g, 4.6 mmol) and triethylamine (0.929292g, 9.2mmol) were added to a 50mL eggplant-shaped bottle containing anhydrous dichloromethane (10 mL) and stirred until dissolved, and ethyl acetoacetate (0.598 g,4.6 mmol) was further added and stirring was continued for 3 hours. The solvent was then removed in vacuo, 9-bromofluorene (1.127g, 4.6 mmol) was added dissolved in DMF (10 mL) and the reaction mixture was stirred for 18h, after which the solution was diluted with ethyl acetate and washed with brine (3 × 15 mL), the combined organic layers were dried over anhydrous magnesium sulfate, concentrated under reduced pressure and separated by silica gel column chromatography (eluent: PE/EtOAc =20/1,15/1,10/1, v/v) to give a white solid (compound 2). Compound 2 was dissolved in acetone (10 mL), and after addition of p-toluenesulfonic acid monohydrate (0.969 g, 5.1mmol) for 1h a white precipitate appeared, which was filtered and the filter cake washed with diethyl ether (5 mL). Finally, the filter cake was dissolved in dichloromethane (20 mL), triethylamine (0.9292g, 9.2mmol) was added and stirred for 2h, further NaOH solution (1m, 10ml) was added to separate the organic phase, the aqueous phase was extracted with dichloromethane, all the organic phases were combined, washed with brine (10 mL), dried and concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =3/1,2/1, v/v) to give a white powder (compound 3, 0.35g, 20%).

(2) Synthesis of Compound CX-1

Bis (triphenylphosphine) palladium dichloride (0.21g, 0.3mmol), benzoyl chloride (1.4g, 10mmol), phenylacetylene (1.276g, 11mmol) and triethylamine (3.5 mL) are added into a 100mL eggplant-shaped bottle filled with 30mL dichloromethane, and finally cuprous iodide (0.19g, 1mmol) is added and reacted for 12 hours under the protection of argon. The reaction mixture was filtered through celite, extracted 3 times with water (3 × 40 mL), the organic layer was dried, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =500/1,400/1,300/1,v/v) to give a tan solid (compound 4). Compound 4 (1.03g, 5 mmol) and ethyl p-aminobenzoate (0.825g, 5 mmol) were sequentially added to a 50mL eggplant-shaped bottle, followed by addition of 10mL of methanol and refluxing at 70 ℃ for 6 hours. The solvent was dried by spinning off and separated by silica gel column chromatography (eluent: PE/EtOAc =30/1,25/1,20/1, v/v) to give a yellow powder (compound 5,1.06g, 57%). Compound 5 (0.371g, 1mmol) and lithium tert-butoxide (0.24g, 3mmol) were sequentially added to a 25mL eggplant-shaped bottle, and 4mL methylene chloride and p-toluenesulfonyl azide (TsN) were added ₃ 0.394g, 2mmol) at room temperature for 12h, detecting by TLC that the reaction was substantially complete, extracting with water (3X 20 mL) and dichloromethane (20 mL) 3 times, drying over anhydrous magnesium sulfate, concentrating under reduced pressureAnd separated by silica gel column chromatography (eluent: PE/EtOAc =20/1,15/1,10/1, v/v) to give a white solid (compound 6,0.23g, yield 78%). Compound 6 (0.1465g, 0.5mmol) was dissolved in 4mL of methanol, followed by dropwise addition of a sodium hydroxide solution (0.2g, 5mmol, dissolved in 0.5mL of water), reaction at room temperature for 12h, completion of the reaction by TLC, adjustment of the pH to about 2 with a 2NHCl solution, extraction with water (20 mL) and ethyl acetate (3X 20 mL) for 3 times, drying over anhydrous magnesium sulfate, and concentration under reduced pressure to obtain a white solid (Compound 7,0.136g, 97% yield). Compound 7 (0.1325g, 0.5mmol) and HATU (0.22g, 0.58mmol) were dissolved in 2mL dmf, DIPEA (0.075g, 0.58mmol) was added dropwise, compound 3 (0.152g, 0.4mmol) was added after reaction at room temperature for 30min, the reaction was overnight, and after completion of the reaction was checked by TLC, extraction was performed 3 times with water (3 × 20 mL) and ethyl acetate (20 mL), and the mixture was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =3/1,2/1, v/v), to obtain a yellow oily liquid (compound 8,0.23g, yield 92%). The compound 8 (0.3135g, 0.5mmol), naHCO ₃ (0.084g, 1mmol) was added to a mixed solution of THF (2 mL) and water (2 mL), and finally Pd/C (1.0 g) was added, the reaction flask was connected with a hydrogen balloon, reacted at room temperature for 12 hours, pd/C was filtered and washed with a mixture of THF (1 mL) and water (1 mL), after adding citric acid (0.126g, 0.6 mmol) to acidify for 30 minutes, water (5 mL) and ethyl acetate (3X 5 mL) were added to extract 3 times, the organic layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: ethyl acetate/petroleum ether =2/1,3/1,1 CH: 2/1,3/1,1 CH ₃ COOH, v/v) to give a pale yellow oily liquid (Compound CX-1,0.095g, yield 40%).

(3) Synthesis of Compound CX-2

Bis (triphenylphosphine) palladium dichloride (0.21g, 0.3mmol), benzoyl chloride (1.4g, 10mmol), phenylacetylene (1.276g, 11mmol) and triethylamine (3.5 mL) are added into a 100mL eggplant-shaped bottle filled with 25mL dichloromethane, and finally cuprous iodide (0.19g, 1mmol) is added and reacted for 12 hours under the protection of argon. The reaction mixture was filtered through celite, extracted 3 times with water (3 × 40 mL), the organic layer was dried, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =500/1,400/1,300/1,v/v) to give a tan solid (compound 4). To a 50mL eggplant-shaped bottle were added compound 4 (1.03g, 5 mmol) and ethyl p-aminobenzoate (0.825g, 5 mmol) in this order) Then, 10mL of methanol was added and the mixture was refluxed at 70 ℃ for 6 hours. The solvent was dried by spinning and separated by silica gel column chromatography (eluent: PE/EtOAc =30/1,25/1,20/1, v/v) to give a yellow powder (compound 5,1.06g, 57%). Compound 5 (0.742g, 2mmol) was dissolved in 5mL of acetonitrile, and p-toluenesulfonyl azide (TsN) was added dropwise ₃ 0.788g, 4mmol) and DBU (0.608g, 4mmol) were reacted at room temperature for 2h, and after completion of the reaction was checked by tlc, the reaction was concentrated under reduced pressure and separated by silica gel column chromatography (eluent: PE/EtOAc =20/1,15/1,10/1, v/v) to give a white powder (compound 9,0.508g, 64% yield). Compound 9 (0.393972 g, 1mmol), sodium borohydride (0.074g, 2mmol) were dissolved in 4mL of ethanol, reacted at room temperature for 4h, after completion of the reaction was checked by TLC, quenched with water, extracted 3 times with ethyl acetate (3X 10 mL), and the organic layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =5/1,4/1,3/1, v/v) to give a white powder (Compound 10,0.356g, 89% yield). Compound 10 (0.2g, 0.5mmol), indole (0.0877g, 0.75mmol) were dissolved in 2mL DCM, and after anhydrous ferric chloride (0.04g, 0.25mmol) was added, the reaction was refluxed at 60 ℃ for 12h, and the reaction was checked for substantial completion, the product was separated by silica gel column chromatography (eluent: PE/EtOAc =10/1,8/1,6/1, v/v), to obtain a white powder (compound 11,0.229g, yield 92%). Compound 11 (0.4 g, 0.8mmol) was dissolved in 4mL of methanol, followed by dropwise addition of a sodium hydroxide solution (0.328 g,8mmol in 0.5mL of water), reaction at room temperature for 12h, completion of the reaction by TLC, adjustment of the pH to about 2 with a 2N Cl solution, extraction with water (20 mL) and ethyl acetate (3X 20 mL) for 3 times, drying of the organic layer over anhydrous magnesium sulfate, and concentration under reduced pressure to give a white solid (Compound 12,0.38g, 97% yield). Compound 12 (0.235g, 0.5 mmol), HATU (0.22g, 0.58mmol) were dissolved in 2mL dmf, DIPEA (0.075g, 0.58mmol) was added dropwise, after reaction at room temperature for 30min, compound 3 (0.152g, 0.4 mmol) was added, the reaction was overnight, after the TLC detection reaction was substantially complete, extracted 3 times with water (3 × 20 mL) and ethyl acetate (20 mL), the organic layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =3/1,2/1, v/v) to give a yellow oily liquid (compound 13,0.316g, yield 95%). Compound 13 (0.25g, 0.3mmol), naHCO ₃ (0.05g, 0.6 mmol) was added to a mixed solution of THF (1.5 mL) and water (1.5 mL), and Pd/C (0.6 g) was finally added thereto, and the reaction flask was connected with a hydrogen balloon at room temperatureReaction for 12h, pd/C was filtered and washed with a mixture of THF (1 mL) and water (1 mL), after adding citric acid (0.084g, 0.4mmol) and acidifying for 30min, water (5 mL) and ethyl acetate (3X 5 mL) were added and extracted 3 times, the organic layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: ethyl acetate/petroleum ether =2/1,3/1,1% CH ₃ COOH, v/v) to give a pale yellow oily liquid (Compound CX-2,0.101g, 50% yield).

(4) Synthesis of Compound CX-3

Bis (triphenylphosphine) palladium dichloride (0.21g, 0.3mmol), benzoyl chloride (1.4g, 10mmol), phenylacetylene (1.276g, 11mmol) and triethylamine (3.5 mL) are added into a 100mL eggplant-shaped bottle filled with 25mL dichloromethane, and finally cuprous iodide (0.19g, 1mmol) is added and reacted for 12 hours under the protection of argon. The reaction solution was filtered through celite, extracted 3 times with water (3 × 40 mL), dried, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =500/1,400/1,300/1,v/v) to give a tan solid (compound 4). Compound 4 (1.03g, 5 mmol) and ethyl p-aminobenzoate (0.825g, 5 mmol) were sequentially added to a 50mL eggplant-shaped bottle, and 10mL of methanol was added thereto, followed by refluxing at 70 ℃ for 6 hours. The solvent was dried by spinning off and separated by silica gel column chromatography (eluent: PE/EtOAc =30/1,25/1,20/1, v/v) to give a yellow powder (compound 5,1.06g, 57%). Compound 5 (0.742g, 2mmol) was dissolved in 5mL of acetonitrile, and p-toluenesulfonylazide (TsN) was added dropwise ₃ 0.788g, 4mmol) and DBU (0.608g, 4mmol) were reacted at room temperature for 2h, and after detection by TLC, the reaction mixture was concentrated under reduced pressure and separated by silica gel column chromatography (eluent: PE/EtOAc =20/1,15/1,10/1, v/v) to give a white powder (compound 9,0.508g, 64% yield). Compound 9 (0.393970 mmol), sodium borohydride (0.074g, 2mmol) were dissolved in 4mL of ethanol and reacted at room temperature for 4h, after TLC detection, quenched with water, extracted 3 times with ethyl acetate (3X 10 mL), dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =5/1,4/1,3/1, v/v) to give a white powder (Compound 10,0.356g, 89% yield). Compound 10 (0.12g, 0.3mmol), 1,3, 5-trimethoxybenzene (0.0756g, 0.45mmol) were dissolved in 2ml of EDCM, and finally anhydrous ferric chloride (0.024g, 0.15mmol) was added, and after refluxing at 60 ℃ for 12h, TLC detection, separation was performed by silica gel column chromatography (eluent: PE/EtOAc =10/1,8/1,6/1, v/v) to obtain a white powder (

compound

14, 0).138g, 84% yield). Dissolving compound 14 (0.1647g, 0.3mmol) in 3mL of methanol, adding dropwise sodium hydroxide solution (0.12g, 3mmol, dissolved in 0.5mL of water), reacting at room temperature for 12h, detecting by TLC, adjusting pH to about 2 with 2NHCl solution, extracting with water (20 mL) and ethyl acetate (3X 20 mL) 3 times, drying, concentrating under reduced pressure, separating by silica gel column chromatography (eluent: PE/EtOAc =3/1,2/1,1 CH) ₃ COOH, v/v) to give a white solid (Compound 15,0.1g, 64% yield). Compound 15 (0.24pg, 0.47mmol), HATU (0.205g, 0.54mmol) were dissolved in 2mL of DMF, DIPEA (0.07g, 0.54mmol) was added dropwise thereto, and after reaction at room temperature for 30min, compound 3 (0.14g, 0.37mmol) was added thereto, the reaction was allowed to proceed overnight, after TLC detection, extraction was performed 3 times with water (3X 20 mL) and ethyl acetate (20 mL), dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: PE/EtOAc =3/1,2/1, v/v) to obtain a yellow oily liquid (Compound 16,0.196g, yield 60%). Mixing compound 16 (0.106g, 0.12mmol), naHCO ₃ (0.02g, 0.24mmol) was added to a mixed solution of THF (1.5 mL) and water (1.5 mL), and finally Pd/C (0.24 g) was added, the reaction flask was connected to a hydrogen balloon, the reaction was carried out at room temperature for 12 hours, the Pd/C was filtered and washed with a mixture of THF (1 mL) and water (1 mL), citric acid (0.042g, 0.2mmol) was added to acidify for 30 minutes, water (5 mL) and ethyl acetate (3X 5 mL) were added to extract 3 times, the organic layer was dried, concentrated under reduced pressure, and separated by silica gel column chromatography (eluent: ethyl acetate/petroleum ether =2/1,3/1,1 CH: (eluent: ethyl acetate/petroleum ether =2/1,3/1,1 CH) ₃ COOH, v/v) to give a pale yellow oily liquid (Compound CX-3,0.045g, yield 52%).

Comparative example 16 protection of the carboxyl groups on APA

1. When the protecting group is 4-methoxybenzyl bromide, compound 3 cannot be formed. The main reasons are: in step iii, no white precipitate was observed in a short time until after about 18 hours, a white precipitate appeared, and the reaction was continued until no product was formed.

2. When the protecting group is diphenylbromomethane, the compound 3 can be produced smoothly. But the yield of compound 3 was very low and almost none.

3. When the protecting group is benzyl bromide, the compound 3 can be generated smoothly, the yield is relatively high and is about 50%, the yield of the amide condensation reaction is also high and is about 85%, but the final product CX-1 cannot be obtained.

The experimental result shows that the protective groups of 4-methoxybenzyl bromide and diphenylbromomethane can not obtain the protected target compound, although the yield of the compound 3 obtained by benzyl bromide protection is higher than that obtained by 9-bromofluorene protection, the yield of the amide condensation product (compound 8) is almost the same, and most importantly, the final product CX-1 can be obtained by 9-bromofluorene protection. Therefore, 9-bromofluorene was selected as the carboxyl protecting agent.

Test example 1

Structural characterization:

9H-fluoren-9yl6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hepta ne-2-carboxylate(3)white solid,mp 170-174℃. ¹ H NMR(400MHz,CDCl ₃ )δ7.66(d,J＝8.0Hz,2H),7.57-7.52(m,2H),7.42(t,J＝8.0Hz,2H),7.31(t,J＝8.0Hz,2H),6.78(s,1H),5.52(d,J＝8.0Hz,1H),4.56(d,J＝8.0Hz,1H),4.51(s,1H),1.60(d,J＝4.0Hz,6H)； ¹³ CNMR(100MHz,CDCl ₃ )δ177.6,168.9,141.1,140.9,129.7,128.0,127.8,126.06,126.01,120.1,120.0,76.3,70.0,64.0,62.8,31.8,27.3.

ethyl 4-(5-phenyl-1H-1,2,3-triazol-1-yl)benzoate(6)white solid,mp 138-141℃. ¹ H NMR(400 MHz,CDCl ₃ )δ8.10(d,J＝8.0 Hz,2H),7.86(s,1H),7.44(d,J＝8.0 Hz,2H),7.40-7.34(m,3H),7.23-7.20(m,2H),4.39(q,J＝8.0 Hz,2H),1.39(t,J＝8.0 Hz,3H)； ¹³ C NMR(100 MHz,CDCl ₃ )δ165.4,139.8,137.8,133.7,130.9,130.6,129.5,129.0,128.6,126.3,124.7,61.4,14.2.

9H-fluoren-9-yl 3,3-dimethyl-7-oxo-6-(4-(5-phenyl-1H-1,2,3-triazol-1-yl)benzamido)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate(8)yellow liquid. ¹ H NMR(400 MHz,CDCl ₃ )δ7.87(d,J＝8.0 Hz,3H),7.68(d,J＝8.0 Hz,2H),7.55(q,J＝8.0 Hz,2H),7.47-7.30(m,9H),7.21(d,J＝8.0 Hz,2H),6.97(d,J＝8.0 Hz,1H),6.8(s,1H),5.66(s,1H),4.59(s,1H),1.63(s,6H)； ¹³ C NMR(100 MHz,CDCl ₃ )δ173.3,168.4,165.3,141.2,141.0,140.8,138.4,137.8,133.7,133.4,129.9,129.5,129.0,128.6,128.4,128.0,127.9,126.2,126.0,125.0,120.2,120.1,76.6,70.6,68.0,65.1,59.0,31.8,27.3.

ethyl 4-(4-benzoyl-5-phenyl-1H-1,2,3-triazol-1-yl)benzoate(9)white solid,mp 140-145℃. ¹ H NMR(400 MHz,CDCl ₃ )δ8.27(d,J＝8.0 Hz,2H),8.08(d,J＝8.0 Hz,2H),7.60(t,J＝8.0 Hz,1H),7.50(t,J＝8.0 Hz,2H),7.44-7.32(m,7H),4.39(q,J＝8.0 Hz,2H),1.39(t,J＝8.0 Hz,3H)； ¹³ C NMR(100 MHz,CDCl ₃ )δ186.5,165.3,143.7,141.2,139.2,137.0,133.2,131.3,130.6,130.1,128.6,128.3,125.7,124.9,61.5,14.2.

ethyl 4-(4-(hydroxy(phenyl)methyl)-5-phenyl-1H-1,2,3-triazol-1-yl)benzoate(10)whitesolid,mp 125-130℃. ¹ H NMR(400 MHz,CDCl ₃ )δ8.04(d,J＝12.0 Hz,2H),7.34-7.28(m,10H),7.11(d,J＝8.0 Hz,2H),5.91(s,1H),4.37(q,J＝8.0 Hz,2H),1.38(t,J＝8.0 Hz,3H)； ¹³ C NMR(100 MHz,CDCl ₃ )δ165.4,147.6,141.9,134.5,130.7,130.5129.8,129.6,128.9,128.4,127.7,126.6,126.1,124.3,68.4,61.4,14.2.

ethyl 4-(4-((1H-indol-3-yl)(phenyl)methyl)-5-phenyl-1H-1,2,3-triazol-1-yl)benzoate(11)yellow solid,mp 181-186℃. ¹ H NMR(400 MHz,CDCl ₃ )δ8.42(s,1H),8.04(d,J＝8.0 Hz,2H),7.47-7.27(m,10H),7.25-7.19(m,2H),7.15-7.12(m,4H),6.98(t,J＝8.0 Hz,1H),5.67(s,1H),4.38(q,J＝8.0 Hz,2H),1.39(t,J＝8.0 Hz,3H)； ¹³ C NMR(100 MHz,CDCl ₃ )δ165.7,148.2,142.7,140.2,136.5,134.0,130.6,130.5,129.9,129.6,129.2,128.7,128.5,127.1,126.7,126.6,124.3,124.1,122.0,119.4,119.1,117.7,111.4,61.5,39.2,14.4.

9H-fluoren-9-yl 6-(4-(4-((1H-indol-3-yl)(phenyl)methyl)-5-phenyl-1H-1,2,3-triazol-1-yl)benzamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate(13)yellow liquid. ¹ H NMR(400 MHz,CDCl ₃ )δ8.33(s,1H),7.76(d,J＝8.0 Hz,2H),7.68(d,J＝8.0Hz,2H),7.56(dd,J ₁ ＝8.0Hz,J ₂ ＝12.0Hz,2H),7.46-7.28(m,15H),7.22-7.11(m,6H),6.98(t,J＝8.0Hz,1H),7.86(d,J＝8.0Hz,1H),6.80(s,1H),5.65(s,1H),4.58(s,1H),1.63(s,6H)； ¹³ C NMR(100MHz,CDCl ₃ )δ173.4,168.4,165.3,148.0,142.5,141.2,141.0,139.6,136.3,133.8,132.9,129.9,129.7,129.5,129.1,128.5,128.4,128.2,128.1,127.9,126.8,126.5,126.0,124.5,123.9,121.9,120.2,120.1,119.3,118.9,117.6,111.2,76.6,70.6,68.0,65.1,59.0,39.0,31.8.

ethyl4-(5-phenyl-4-(phenyl(2,4,6-trimethoxyphenyl)methyl)-1H-1,2,3-triazol-1-yl)benzoate(14)white solid,mp 197-201℃. ¹ H NMR(400MHz,CDCl ₃ )δ8.00(d,J＝8.0Hz,2H),7.37(d,J＝8.0Hz,2H),7.31-7.28(m,3H),7.21(q,J＝4.0Hz,4H),7.12(t,J＝8.0Hz,1H),6.96(d,J＝8.0Hz,2H),6.06(s,1H),6.04(s,2H),4.36(q,J＝8.0Hz,2H),3.76(s,3H),3.54(s,6H),1.37(t,J＝8.0Hz,3H)； ¹³ C NMR(100MHz,CDCl ₃ )δ165.5,160.1,158.8,147.4,142.3,140.3,134.2,130.3,130.0,129.7,128.6,128.5,128.2,127.6,127.5,125.5,124.0,111.8,91.2,61.2,55.5,55.1,36.7,14.2.

9H-fluoren-9-yl3,3-dimethyl-7-oxo-6-(4-(5-phenyl-4-(phenyl(2,4,6-trimeth-oxyphenyl)methyl)-1H-1,2,3-triazol-1-yl)benzamido)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate(16)yellow liquid. ¹ H NMR(400MHz,CDCl ₃ )δ7.74(d,J＝8.0Hz,2H),7.68(d,J＝8.0Hz,2H),7.56(dd,J ₁ ＝8.0Hz,J ₂ ＝12.0Hz,2H),7.44(t,J＝8.0Hz,2H),7.39(d,J＝8.0Hz,2H),7.34-7.28(m,5H),7.21(t,J＝8.0Hz,4H),7.12(t,J＝8.0Hz,1H),6.95(d,J＝8.0Hz,2H),6.82(d,J＝12.0Hz,2H),6.05(s,1H),6.03(s,2H),5.64(s,1H),4.58(s,1H),3.76(s,3H),3.53(s,6H),1.63(s,6H)； ¹³ C NMR(100MHz,CDCl ₃ )δ173.4,168.4,165.4,160.2,158.8,147.5,142.3,141.2,141.0,140.8,139.9,134.2,132.5,129.9,129.7,128.7,128.5,128.3,128.1,127.9,127.6,126.0,125.5,124.4,120.2,120.1,111.7,91.2,76.6,70.6,68.1,65.1,58.9,55.5,55.2,36.7,31.8,27.3.

determination of Minimum Inhibitory Concentration (MIC) of Compounds

Mueller hinton liquid medium: 0.24g of dry MH (B) medium was dissolved in 10mL of distilled water, and 2.4g of dry MH (B) medium was dissolved in 100mL of distilled water.

Preparation of sample liquid and positive liquid

Sample liquid medicine: accurately weighing samples CX-1 (5.93 mg), CX-2 (8.56 mg) and CX-3 (9.21 mg) in a 1.5mLep tube, adding 1mLDMSO to completely dissolve the samples to obtain a mother solution with the concentration of 12.8mM, and then carrying out double dilution on the mother solution by using a liquid culture medium to obtain the following concentration: 256,128,64,32,16,8,4,2,1,0.5 and 0.25 mu M of liquid medicine for standby.

Positive liquid medicine: accurately weighing 0.29mg of ampicillin sodium (Amp), dissolving in 6mL of liquid culture medium to obtain a mother solution with the concentration of 128 mu M, and performing twofold dilution on the mother solution with the liquid culture medium to obtain a solution with the concentration of: 64,32,16,8,4,2,1,0.5,0.25,0.125 and 0.0625 μ M of the liquid medicine is reserved for use.

Preparation of bacterial liquid

Under aseptic condition, taking single colony in sterilized 10mL liquid culture medium, shaking at 37 deg.C for 200r/min, culturing for 24h, and determining its OD _600nm Then, the resulting suspension was diluted 1000-fold with a medium to a bacterial solution concentration of 1.0X 10 ⁵ CFU/mL is ready for use.

Determination of samples

Sequentially adding 50 mu L of bacterial liquid into the 1 st to 11 th columns on a 96-well plate, and sequentially adding sample solutions with different concentrations from 1 to 11 from large to small (128, 64,32,16,8,4,2,1,0.5,0.25 and 0.125 mu M) into the 1 st to 11 th columns in the A to D rows; the positive drug solutions with different concentrations are added in sequence from the 1 st column to the 11 th column from the E th column to the H th column, and the concentrations are from 1 to 11 and are gradually decreased (32, 16,8,4,2,1,0.5,0.25,0.125,0.0625,0.03125 mu M); in the first four wells of column 12, 50. Mu.L of the cell suspension and 50. Mu.L of the culture medium (negative control without drug) were added, and in the second four wells, 100. Mu.L of the culture medium (blank control) was added. After culturing for 24h in a biochemical incubator at 37 ℃, the results are observed.

Results and discussion

The results of the experiment are shown in table 1:

TABLE 1 MIC (μ M) of samples against 3 bacteria

Note: 1. visually observing the turbid front hole to obtain the MIC value of the compound; 2. when the MIC value is read, if the 11 th hole is clear, the dilution is continued until the previous hole which is turbid is read as the MIC value; if the 1 st well is turbid, it is recorded as > 128. Mu.M.

After a period of culture, the negative control group of each 96-well plate has bacteria growing normally, the blank control group has no bacteria growing, and the results after three times of experiments are the same, so the experimental results are credible. As can be seen from Table 3, compounds CX-1, CX-2 and CX-3 inhibit the growth of Staphylococcus aureus and Streptococcus mutans, but they are inferior to ampicillin, which is a positive drug. Wherein, CX-2 has similar inhibition effect on the two bacteria, MIC is 4 mu M, CX-1 has better inhibition effect on staphylococcus aureus than Streptococcus mutans, and CX-3 is just good and opposite; however, none of the 3 compounds tested had inhibitory activity against E.coli.

Determination of beta-lactamase Activity of Compounds

Experimental Material

Apparatus and equipment: multifunctional microplate readers (SynergyHTX), gene ltd; microplate constant temperature incubator (model VS 60-4F), wuxi WooXin instruments Ltd.

Reagent: nitrocefin (ab 145625), abcam corporation; recombinant Acinetobacter Beta-lactamase OXA-23 (230-00712-50), ray Biotech; DMSO, aladine; HEPES (1112 GR 100), biofroxx Germany; sodium chloride (BC 13), guangzhou chemical reagent plant; bovine Serumalbumin (B2064), sigma-Aldrich; triton X-100 (0694), amresco Inc.

Principle of experiment

The cephalonitrothiophene is a chromogenic beta-lactamase substrate, and when an amide bond in a beta-lactam ring is hydrolyzed by the beta-lactamase, the color changes from yellow to red. The cephalonitrothiophene with the complete structure is positioned in an ultraviolet light absorption area (about 380 nm), and an enzymolysis product of the cephalonitrothiophene is transferred to a visible light spectrum area (about 500 nm). Therefore, the content of the enzymolysis product can be obtained by detecting the absorbance at 492nm, and the percentage inhibition rate of the compound to the enzyme can be calculated.

Experimental methods

Preparation of stock solution

Preparation of sample solution: compound CX-1 was diluted from the stock solution at 12.8mM to a solution of 8. Mu.M, 4. Mu.M, 2. Mu.M, 1. Mu.M, 100nM, 10nM, 1nM for use. Then compounds CX-2 and CX-3 were diluted to 100nM solution for further use.

Preparation of 1 × kinase buffer: 20mM HEPES buffer, 0.25M NaCl solution, 1. Mu.g/ml BA, 0.01% Triton X-100%.

Preparing a cefuroxime sodium solution: 1.033mg of cefuroxime axetil was dissolved in DMSO, and a1 Xkinase buffer solution was added thereto at a concentration of 2mM.

Preparation of 4 xOXA-23: the stock solution was 50. Mu.g/mL OXA-23 diluted to 1.2. Mu.g/mL mLOXA-23 with 1 Xkinase buffer for use.

Determination of Compound Activity

50. Mu.L of a compound CX-1, 1xkinase buffer solution at a concentration of 8. Mu.M, 4. Mu.M, 2. Mu.M, 1. Mu.M, 100nM, 10nM, 1nM was added in the 96-well plate in the order of line 1 to line 8, line A to line C; then adding 1.2 mu g/mLOXA-23, 25 mu L of each hole, and incubating for 10min at 37 ℃; adding 2mM of cefuroxime sodium, incubating at 37 ℃ for 30min, wherein each well is 25 mu L; the absorbance was measured at 492nm, and the inhibition ratio was calculated. The compounds CX-2 and CX-3 were used at a concentration of 100nM as described above, and the absorbance at 492nM was measured to calculate the inhibition ratio.

Data processing

The inhibition rate of the compound on enzyme is calculated by the following formula:

inhibition ratio (%) = (1-NHC _{Sample (I)} /NHC _{Negative of} )×100

NHC: hydrolysis constant of cefuroxime

NHC _{Negative of} : average hydrolysis constant of all negative controls on plate (1 Xkinase buffer)

Results and discussion

TABLE 2 inhibition of beta-lactamase by samples of different concentrations

The experimental results are shown in table 42, and firstly, we measured the activity of inhibiting beta-lactamase for CX-1 with different concentrations based on the experiment of the minimum inhibitory concentration, and the results show that: when the concentration is 4 muM, 0.1 muM and 0.01 muM, CX-1 has inhibition effect on beta-lactamase, the best is 0.1 muM, and the inhibition rate is 15.53%; when compounds CX-2 and CX-3 were measured at a concentration of 0.1. Mu.M, the inhibition rates were 7.16% and 0.73%, respectively, and as a result, they were not similar to the compound CX-1, and CX-3 had substantially no inhibition effect.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A synthetic method of triazole penicillin molecules is characterized in that a compound CX-1 or/and a compound CX-2 or/and a compound CX-3 are synthesized after protection of carboxyl on 6-aminopenicillanic acid, wherein the structural formula of the compound CX-1 is shown as a formula I, the structural formula of the compound CX-2 is shown as a formula II, and the structural formula of the compound CX-3 is shown as a formula III:

2. the method for synthesizing triazole penicillin molecules as claimed in claim 1, wherein the step of protecting carboxyl groups on 6-aminopenicillanic acid comprises the steps of mixing 6-aminopenicillanic acid, triethylamine and ethyl acetoacetate for reaction, adding 9-bromofluorene and N, N-dimethylformamide for reaction, drying, concentrating, separating eluent 1 through silica gel column chromatography to obtain compound 2, dissolving compound 2 in acetone, adding p-toluenesulfonic acid monohydrate, filtering to obtain filter cake, dissolving the filter cake in dichloromethane, adding triethylamine, separating an organic phase by using sodium hydroxide solution, extracting a water phase with dichloromethane, combining the organic phases, washing the organic phase, drying, concentrating under reduced pressure, and separating eluent 2 through silica gel column chromatography to obtain compound 3, wherein structural formulae IV of compound 2 and compound 3 are shown as formula IV:

3. the method for synthesizing molecules containing triazole penicillin as claimed in claim 2, wherein the eluent 1 is a mixture of 10-20:1, and an eluent 2 is petroleum ether and ethyl acetate, wherein the volume ratio of the eluent 2 is 2-3:1 petroleum ether and ethyl acetate.

4. The method for synthesizing molecules containing triazole penicillin as claimed in claim 2, wherein the mass ratio of 6-aminopenicillanic acid, triethylamine, ethyl acetoacetate, 9-bromofluorene and toluenesulfonic acid monohydrate is 0.95-1.05.

5. The method for synthesizing triazole penicillin molecules as claimed in claim 1, wherein said compound CX-1 is synthesized by sequentially adding palladium bis (triphenylphosphine) dichloride, benzoyl chloride, phenylacetylene and triethylamine into a container filled with dichloromethane, finally adding cuprous iodide, reacting under the protection of inert gas to obtain compound 4, mixing compound 4 with ethyl p-aminobenzoate and methanol, and extracting under reflux to obtain compound 5; sequentially adding the compound 5 and lithium tert-butoxide into an eggplant-shaped bottle, and then adding dichloromethane and p-toluenesulfonyl azide for reaction to obtain a compound 6; dissolving the compound 6 in methanol, dropwise adding a sodium hydroxide solution for reaction, and adjusting the pH value by using a hydrochloric acid solution to obtain a compound 7; dissolving a compound 7 and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate in N, N-dimethylformamide, adding N, N-diisopropylethylamine for reaction, then adding a compound 3 for reaction to obtain a compound 8, dissolving the compound 8 and sodium bicarbonate in a tetrahydrofuran mixed solution, adding a catalyst for reaction to obtain a compound CX-1, wherein the synthetic route of the compound CX-1 is shown as a formula V:

6. the method for synthesizing triazole penicillin molecules as claimed in claim 1, wherein the mass ratio of bis (triphenylphosphine) palladium dichloride, benzoyl chloride, phenylacetylene, triethylamine and cuprous iodide is 0.19-23; the mass ratio of the compound 4 to the ethyl p-aminobenzoate to the methanol is 1-1.05; the mass ratio of the compound 5, lithium tert-butoxide, dichloromethane and p-toluenesulfonyl azide is 0.37-0.38; the mass ratio of the compound 6 to the methanol to the sodium hydroxide solution is 0.14-0.15; the mass ratio of the compound 7, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate to N, N-dimethylformamide to N, N-diisopropylethylamine to the compound 3 is 0.13-0.135; the mass ratio of the compound 8, sodium bicarbonate and tetrahydrofuran mixed solution is 0.31-0.32, the pH value is adjusted to 1.8-2.2 by using 1-2mol/L hydrochloric acid solution, the tetrahydrofuran mixed solution is tetrahydrofuran and water with the volume ratio of 1.

7. The method for synthesizing triazole penicillin molecules according to claim 1, wherein the compound CX-2 is synthesized by dissolving compound 5 in acetonitrile, adding p-toluenesulfonyl azide and 1, 8-diazabicycloundec-7-ene to react, preparing compound 9, dissolving compound 9 and sodium borohydride in ethanol to react, preparing compound 10, dissolving compound 10 and indole in dichloromethane, adding anhydrous ferric chloride to carry out reflux reaction, preparing compound 11, dissolving compound 11 in methanol, dropwise adding sodium hydroxide solution to react, dissolving hydrochloric acid in a pH-adjusted solution to carry out extraction, preparing compound 12, dissolving compound 12 and 2- (7-azabenzotriazole) -N, N' -tetramethylurea hexafluorophosphate in N, N-dimethylformamide, adding N, N-diisopropylethylamine to react, adding compound 3 to react, preparing compound 13, dissolving compound 13 and sodium bicarbonate in a tetrahydrofuran mixed solution, adding a catalyst to react, preparing compound CX-2, and the synthetic route vi of compound CX-2 is shown in formula vi:

8. the method for synthesizing molecules containing triazole penicillin as claimed in claim 7, wherein the mass ratio of the compound 5, acetonitrile, p-toluenesulfonyl azide and 1, 8-diazabicycloundecen-7-ene is 0.74-0.75; the mass ratio of the compound 9 to the sodium borohydride to the ethanol is 0.39-0.40; the mass ratio of the compound 10 to the indole to the dichloromethane to the anhydrous ferric chloride is 0.15-0.25; the mass ratio of the compound 11 to the methanol to the sodium hydroxide solution is 0.3-0.5; the mass ratio of the compound 12, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate to N, N-dimethylformamide to N, N-diisopropylethylamine to the compound 3 is 0.23-0.24; the mass ratio of the compound 13, sodium bicarbonate and tetrahydrofuran mixed solution is 0.2-0.3, the catalyst is Pd/C, the pH value is adjusted to 1.8-2.2 by using 1-2mol/L hydrochloric acid solution, and the tetrahydrofuran mixed solution is tetrahydrofuran and water in a volume ratio of 1.

9. The method for synthesizing triazole penicillin molecules as claimed in claim 1, wherein the synthesis of the compound CX-3 comprises the steps of dissolving compound 10 and 1,3, 5-trimethoxybenzene in dichloromethane, adding anhydrous ferric chloride, carrying out reflux reaction to obtain compound 14, dissolving compound 14 in methanol, dropwise adding sodium hydroxide solution for reaction, dissolving hydrochloric acid for adjusting the pH value, carrying out extraction to obtain compound 15, dissolving compound 15 and 2- (7-azabenzotriazole) -N, N' -tetramethylurea hexafluorophosphate in N, N-dimethylformamide, adding N, N-diisopropylethylamine for reaction, adding compound 3 for reaction to obtain compound 16, dissolving compound 16 and sodium bicarbonate in tetrahydrofuran mixed solution, adding a catalyst for reaction to obtain compound CX-3, and the synthetic route of compound CX-3 is shown as formula vii:

10. the method for synthesizing molecules containing triazole penicillin as claimed in claim 9, wherein the mass ratio of the compound 10, 1,3, 5-trimethoxybenzene, dichloromethane and anhydrous ferric chloride is 0.1-0.14; the mass ratio of the compound 14 to the methanol to the sodium hydroxide solution is (0.16-0.17); the mass ratio of a compound 15, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate to N, N-dimethylformamide to N, N-diisopropylethylamine to a compound 3 is (0.24-0.25); the mass ratio of the compound 16, the sodium bicarbonate and the tetrahydrofuran mixed solution is 0.1-0.11, the catalyst is Pd/C, and 1-2mol/L hydrochloric acid solution is used for adjusting the pH value to 1.8-2.2; the tetrahydrofuran mixed solution is tetrahydrofuran and water with the volume ratio of 1.