WO2017113525A1 - Application of n-benzylimidazolecarboxamide derivatives as synergist for polymyxin antibiotics - Google Patents

Application of n-benzylimidazolecarboxamide derivatives as synergist for polymyxin antibiotics Download PDF

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WO2017113525A1
WO2017113525A1 PCT/CN2016/077918 CN2016077918W WO2017113525A1 WO 2017113525 A1 WO2017113525 A1 WO 2017113525A1 CN 2016077918 W CN2016077918 W CN 2016077918W WO 2017113525 A1 WO2017113525 A1 WO 2017113525A1
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benzylmidamide
derivative
polymyxin
imidazole
use according
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PCT/CN2016/077918
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French (fr)
Chinese (zh)
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彭险峰
覃宗华
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广州英赛特生物技术有限公司
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Publication of WO2017113525A1 publication Critical patent/WO2017113525A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/116—Heterocyclic compounds
    • A23K20/137—Heterocyclic compounds containing two hetero atoms, of which at least one is nitrogen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/195—Antibiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4174—Arylalkylimidazoles, e.g. oxymetazolin, naphazoline, miconazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods

Definitions

  • the invention belongs to the field of biomedicine and relates to the application of N-benzylmidamide derivatives as synergistic synergists of polymyxin antibiotics, such as preparation of human drugs and application as an animal control agent and feed additive.
  • Polymyxin is an antibacterial polypeptide found in Bacillus polymyxa culture medium. It has five kinds of A, B, C, D, E, etc. It is similar in structure, and the antibacterial spectrum is similar to each other and has a wide range. It has a killing effect on Gram-negative bacteria in particular. Polymyxin B and polymyxin E (colistin sulfate) are commonly used in clinical practice. Due to its high toxicity, it is mainly used for local treatment of sensitive bacteria. Infection of Pseudomonas aeruginosa in eyes, ears, skin, mucosal infections and burns and oral administration for preoperative preparation of the intestines. As a feed additive application in the feed industry, it can prevent livestock diseases, improve the metabolism of livestock and poultry, and increase the survival rate and feed conversion rate to promote the growth of livestock and poultry.
  • One of the objects of the present invention is to provide a novel, safe and effective synergistic synergist for polymyxins.
  • the colistin synergist provided by the present invention is an N-benzylmidamide derivative.
  • the invention therefore provides for the use of a N-benzylmidamide derivative as a synergistic synergist for a polymyxin antibiotic having a structure as shown in formula (I):
  • the N- benzyl-imidazole derivatives R 1 is preferably H, R 2 is preferably halogen.
  • N- benzyl-imidazole derivatives R 1 is preferably H, R 2 is preferably Cl.
  • N-benzylmidamide derivative is preferably a compound of formula (II),
  • polymyxins of the present invention are selected from the group consisting of polymyxin A, polymyxin B, polymyxin C, polymyxin D or polymyxin E.
  • the N-benzylmidamide derivative provided by the invention has a remarkable synergistic effect on the inhibition of bacterial growth by colistin sulfate in an in vitro antibacterial test research process, and the minimum inhibitory concentration of colistin sulfate in the corresponding experiment There was a significant drop.
  • the test is separately in the culture When the N-benzylmidamide derivatives containing the same concentration of different structures were used, the minimum inhibitory concentration of polymyxin antibiotics on the corresponding sensitive strains in vitro, N-benzylmidamide derivatives showed obvious synergistic synergistic effect.
  • the minimum inhibitory concentration of the polymyxin antibiotic to the corresponding susceptible strain in the same concentration of the different concentration of the N-benzylmidamide derivative in the culture is tested, and the preferred test susceptible strain is Polymyxin-like antibiotic-sensitive E. coli.
  • synergistic effects of different concentrations of the same N-benzylmidamide derivative on polymyxin antibiotic resistant strains exhibit a dose effect.
  • the bacteria are Gram-negative bacteria, including Escherichia coli, Enterobacter, Pseudomonas aeruginosa, Shigella, Klebsiella, Salmonella, Clostridium perfringens, Staphylococcus aureus and the like.
  • a second object of the present invention is to provide the use of the N-benzylmidamide derivative as a polymyxin antibiotic synergist for the preparation of a medicament for antibacterial infection in animals, such as for preparation A drug that is resistant to polymyxin antibiotic-sensitive or resistant bacteria.
  • the common bacterial infection symptoms of animals are bacterial diarrhea caused by intestinal infection, which is characterized by diarrhea, loss of appetite, indigestion, mental dysfunction, body weight loss and death. Bacterial diarrhea is contagious and can affect the health of other livestock in the same group. Polymyxins have therapeutic and preventive effects on bacterial sputum in animals.
  • the polymyxin antibiotic synergist N-benzylmidamide derivative of the present invention is administered orally in an oral dosage form.
  • the oral dosage form comprises, in addition to the main drug component N-benzylimimidamide derivative, a pharmaceutically acceptable carrier, excipient, diluent, antioxidant, anti-caking agent, vehicle or a combination thereof.
  • the therapeutic oral dosage forms are tablets, capsules, granules, powders, oral liquids, powders, premixes and the like.
  • polymyxin antibiotic synergist of the present invention is prepared as a compound preparation in combination with a polymyxin antibiotic.
  • the compound preparation can be used orally, and contains a pharmaceutically acceptable carrier, an excipient, a diluent, an antioxidant, an anti-caking agent in addition to the N-benzylmidamide derivative and the polymyxin antibiotic. , solvent or a combination thereof.
  • the compound preparations are tablets, capsules, granules, powders, oral liquids, powders, premixes and the like.
  • the N-benzylmidamide derivative is used in a therapeutic dose of from 1 to 200 ppm, preferably from 10 to 200 ppm.
  • the N-benzylmidamide derivative is used as a polymyxin antibiotic synergist for anti-animal bacterial infection, and the animal is mainly poultry and livestock, specifically including chicken, duck, goose, pigeon, donkey, pig, Cow, sheep, horse, rabbit, donkey, dog, cat, fox, donkey or donkey.
  • a third object of the present invention is to provide the use of the N-benzylmidamide derivative as a polymyxin antibiotic synergist for the preparation of an animal growth promoter.
  • Polymyxin antibiotics have a significant effect on improving the performance of animals in the feed processing industry as an antibiotic animal growth promoter.
  • the N-benzylmidamide derivatives are compatible with polymyxin antibiotics. Synergistically enhances the effect of promoting animal growth performance.
  • the N-benzylmidamide derivative is formulated with a feed carrier, diluent, antioxidant, anti-caking agent, vehicle, or a combination thereof to produce a feed additive for growth promotion in an animal.
  • the N-benzylmidamide derivative and the polymyxin antibiotic are combined with a feeding carrier, diluent, antioxidant, anti-caking agent, vehicle, or a combination thereof to form a compound feed additive for use in The growth of animals.
  • the feed additive is a tablet, a powder, a powder, a granule, an oral solution, a capsule, a premix, and the like.
  • the feed additive provided by the present invention is added to the animal's daily ration in a separate form.
  • the feed additive, the feed additive, the carrier or the diluent provided by the present invention are prepared into an additive premixed feed, concentrated feed, compound feed, concentrate supplement feed, etc. in a certain ratio for the animal. edible.
  • the feed additive is used in the feed at a dose of N-benzylmidamide derivative in an amount of from 1 to 1000 ppm, preferably from 10 to 500 ppm.
  • N-benzylmidamide derivatives are used as synergistic synergists for polymyxins in animals for growth.
  • the animals are mainly poultry and livestock, including chickens, ducks, geese, pigeons, quails, pigs, and cattle. , sheep, horses, rabbits, donkeys, dogs, cats, foxes, baboons or baboons.
  • the present invention also provides a synergistic synergist of an N-benzylmidamide derivative as a polymyxin antibiotic for the preparation of a medicament for human infection against Gram-negative bacteria.
  • polymyxin antibiotics are highly toxic, and has adverse reactions such as nervous system toxicity and allergic reactions such as itching, fever and rash, which limits the clinical use and utilization rate of the drug.
  • the N-benzylmidamide derivative has synergistic effect on polymyxin antibiotics, and the combined use thereof can significantly reduce the dosage of polymyxin antibiotics and reduce the probability and intensity of toxic side reactions.
  • the N-benzylmidamide derivative of the present invention is combined with polymyxin antibiotics in the preparation of a compound therapeutic drug for human use Applications.
  • the compound therapeutic drug comprises one of N-benzylimimidamide derivative and polymyxin antibiotic and other medicinal excipients, and may be powder, granule, tablet, capsule, ointment, eye ointment, gel Agents, aerosols, films, oral liquids, eye drops, injections, suppositories, and the like.
  • the compound therapeutic drug can be used for treating diseases caused by Gram-negative bacteria infection such as gastrointestinal infection, sepsis, urinary tract infection, dysentery, whooping cough, respiratory tract infection, biliary tract infection, burn or traumatic wound infection caused by human bacteria.
  • the N-benzylmidamide derivative provided by the invention can be used as a novel effective and safe polymyxin antibiotic preparation for animal disease prevention and treatment, and as a synergistic agent to improve the growth of so-called antibiotic animals.
  • the use efficiency of the accelerator thereby improving the production performance of the animal improves the economic benefit of the feeding, and can reduce the dosage of the polymyxin antibiotic in the clinical use of the human medicine, thereby reducing the occurrence of toxic side reactions and improving the use efficiency of the medicine.
  • the invention also relates to methods and processes for the preparation, isolation, purification, and the like of the N-benzylmidamide derivatives.
  • any of the embodiments of any of the aspects of the invention may be combined with other embodiments as long as no contradiction occurs between them.
  • any of the technical features may be applied to the technical features in other embodiments as long as there is no contradiction between them.
  • C 1-4 straight or branched alkyl means methyl, ethyl, propyl, isopropyl, C 4 alkyl straight or branched An alkane group
  • C 1-4 straight or branched haloalkyl represents a methyl, ethyl, propyl, isopropyl, C 4 alkyl straight or branched alkane group and the alkane group is at least one halogen atom
  • OC 1-4 represents an alkoxy group
  • substituted means that one or more hydrogen atoms in a given structure are replaced by a particular substituent.
  • a substituted group may have one or more substituents substituted at each substitutable position of the group. When more than one position in the given formula can be substituted by one or more substituents selected from a particular group, The substituents may then be substituted at the same or different positions.
  • linking substituents are described.
  • the Markush variable recited for that group is understood to be a linking group.
  • the Markush group definition for the variable recites “alkyl”, it should be understood that the "alkyl” represents a linked alkylene group.
  • N-benzylmidamide derivatives of the present invention can be prepared by the methods described herein, unless otherwise stated, wherein the substituents are as defined for formula (I).
  • the following reaction schemes and examples are provided to further illustrate the contents of the present invention.
  • Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (9.2 g, 50 mmol, 1 eq), 2-chloro-6-fluorobenzyl chloride (17.9 g, 100 mmol, 2 eq) and potassium carbonate (17.3 g, 125 mmol, 2.5 eq) was dissolved in 70 mL of N,N-dimethylformamide, and the reaction was stirred at 100 ° C for 15 h, and the reaction was monitored by TLC to the end.
  • reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL ⁇ 3), and the ethyl acetate layer was combined, washed with water (100 mL ⁇ 3) and brine (100 mL ⁇ 2), dried over anhydrous sodium sulfate.
  • the organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(2-chloro-6fluorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester), which was directly used for the next reaction.
  • the product obtained in the first step 1-(2-chloro-6fluorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester, is dissolved in about 50 ml of aqueous ammonia, and stirred at 45 ° C overnight to obtain a white suspension. After filtration, the filter cake was washed with water (20 mL ⁇ 3), and dried under reduced pressure at 55 ° C to give 11.1 g of the product (1-(2-chloro-6-fluorobenzyl)-1H-imidazole-4,5-diamide). The total yield was 79.8%.
  • the reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL ⁇ 3), and the ethyl acetate layer was combined, washed with water (100 mL ⁇ 3) and brine (100 mL ⁇ 2), dried over anhydrous sodium sulfate.
  • the organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(4-chlorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
  • the reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL ⁇ 3), and the ethyl acetate layer was combined, washed with water (100 mL ⁇ 3) and brine (100 mL ⁇ 2), dried over anhydrous sodium sulfate.
  • the organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(3-chlorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
  • reaction solution was cooled to room temperature, added with 150 mL of water and ethyl acetate (100 mL) ⁇ 3), the ethyl acetate layer was combined, washed with water (100 mL ⁇ 3) and brine (100 mL ⁇ 2), dried over anhydrous sodium sulfate, and evaporated.
  • (4-Fluorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester was used directly in the next reaction.
  • Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (6.0 g, 32.6 mmol, 1 eq), p-nitrobenzyl chloride (11.2 g, 65.2 mmol, 2 eq) and potassium carbonate (11.3 g, 81.5 mmol, 2.5 Eq) was dissolved in 50 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 15 h, and the reaction was monitored by TLC to the end.
  • the reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL ⁇ 3), and the ethyl acetate layer was combined, washed with water (100 mL ⁇ 3) and brine (100 mL ⁇ 2), dried over anhydrous sodium sulfate.
  • the organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(4-nitrobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
  • the product obtained in the first step is obtained by dissolving the product of 1-(4-nitrobenzyl)-1H-imidazole-4,5-dicarboxylate in about 50 ml of aqueous ammonia, and stirring at 45 ° C overnight to obtain a white suspension, which is filtered.
  • the filter cake was washed with water (20 mL ⁇ 3), and dried under reduced pressure at 55 ° C to give 4.8 g of the product (1-(4-nitrobenzyl)-1H-imidazole-4,5-diamide). The total yield was 50.5%.
  • Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (7.0 g, 38.0 mmol, 1 eq), p-methylbenzyl chloride (10.7 g, 76.0 mmol, 2 eq) and potassium carbonate (13.1 g, 95.0 mmol, 2.5 Eq) was dissolved in 50 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 15 h, and the reaction was monitored by TLC to the end.
  • the reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL ⁇ 3), and the ethyl acetate layer was combined, washed with water (100 mL ⁇ 3) and brine (100 mL ⁇ 2), dried over anhydrous sodium sulfate.
  • the organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(4-methylbenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
  • the product obtained in the first step is obtained by dissolving the product of 1-(4-methylbenzyl)-1H-imidazole-4,5-dicarboxylate in about 50 ml of aqueous ammonia, and stirring at 45 ° C overnight to obtain a white suspension, which is filtered. The filter cake was washed with water (20 mL ⁇ 3), and dried under reduced pressure at 55 ° C to give y. The total yield was 79%.
  • Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (7.0 g, 38.0 mmol, 1 eq), p-trifluoromethylbenzyl bromide (18.2 g, 76.0 mmol, 2 eq) and potassium carbonate (13.1 g, 95.0 mmol) , 2.5 eq) was sequentially dissolved in 50 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 15 h, and the reaction was monitored by TLC to the end.
  • the reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL ⁇ 3), and the ethyl acetate layer was combined, washed with water (100 mL ⁇ 3) and brine (100 mL ⁇ 2), dried over anhydrous sodium sulfate.
  • the organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(4-trifluoromethylbenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
  • the product obtained in the first step 1-(4-trifluoromethylbenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester, is dissolved in about 50 ml of aqueous ammonia, and stirred at 45 ° C overnight to obtain a white suspension. After filtration, the filter cake was washed with water (20 mL ⁇ 3), and dried under reduced pressure at 55 ° C to give the product (1-(4-trifluoromethylbenzyl)-1H-imidazole-4,5-diamide) 8.73 g. The total yield was 73%.
  • Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (9.2 g, 50 mmol, 1 eq), p-methoxybenzyl chloride (15.7 g, 100.0 mmol, 2 eq) and potassium carbonate (17.3 g, 125.0 mmol, 2.5 Eq) was dissolved in 70 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 15 h, and the reaction was monitored by TLC to the end.
  • the reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL ⁇ 3), and the ethyl acetate layer was combined, washed with water (100 mL ⁇ 3) and brine (100 mL ⁇ 2), dried over anhydrous sodium sulfate.
  • the organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(4-methoxybenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
  • the product obtained in the first step is obtained by dissolving the product of 1-(4-methoxybenzyl)-1H-imidazole-4,5-dicarboxylic acid in about 50 ml of aqueous ammonia, and stirring at 45 ° C overnight to obtain a white suspension.
  • the filter cake was washed with water (20 mL ⁇ 3), and dried under reduced pressure at 55 ° C to give the product (1-(4-methoxybenzyl)-1H-imidazole-4,5-diamide) 8.55 g.
  • the total yield was 62%.
  • Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (9.2 g, 50 mmol, 1 eq), 3,4-dichlorobenzyl bromide (24.0 g, 100.0 mmol, 2 eq) and potassium carbonate (17.3 g, 125.0 mmol, 2.5 eq) were sequentially dissolved in 70 mL of N,N-dimethylformamide, and the reaction was stirred at 110 ° C for 14 h, and the reaction was monitored by TLC to the end.
  • the reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL ⁇ 3), and the ethyl acetate layer was combined, washed with water (100 mL ⁇ 3) and brine (100 mL ⁇ 2), dried over anhydrous sodium sulfate.
  • the organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(3,4-dichlorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
  • the product obtained in the first step 1-(3,4-dichlorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester, is dissolved in about 50 ml of aqueous ammonia, and stirred at 45 ° C overnight to obtain a white suspension. After filtration, the filter cake was washed with water (20 mL ⁇ 3), and dried under reduced pressure at 55 ° C to give 11.1 g of the product (1-(3,4-dichlorobenzyl)-1H-imidazole-4,5-diamide). The total yield is 75%.
  • 3,5-Dinitrobenzoic acid (12 g, 56.6 mmol, 1 eq) was dissolved in 100 ml of tetrahydrofuran. After cooling to 0 ° C, sodium borohydride (4.3 g, 113.2 mmol, 2.0 eq) was added, and the reaction was stirred for 1 h, slowly added dropwise. Boron trifluoride.
  • 3,5-Dinitrobenzyl alcohol (2.4 g, 12.12 mmol, 1 eq) was dissolved in dry 50 mL dichloromethane, cooled to 0 ° C, triethylamine (4.9 g, 48.2 mmol, 4 eq) Sulfoxide (2.9 g, 24.2 mmol, 2 eq) was added dropwise, gradually warmed to reflux, reacted for 3 h, then the mixture was cooled and cooled to room temperature, and 50 mL of water was added dropwise to quench the reaction and the layers were separated. The organic layer was dried over anhydrous sodium sulfate and evaporated, evaporated, evaporated, evaporated. Dinitrobenzyl chloride) 2.5 g, yield 95.3%.
  • the reaction mixture was cooled to room temperature, and 15 mL of water was added, and ethyl acetate (10 mL ⁇ 3) was added, and the ethyl acetate layer was combined, washed with water (10 mL ⁇ 3) and brine (10 mL ⁇ 2) and dried over anhydrous sodium sulfate.
  • the organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(3,5-dinitrobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
  • the product obtained in the step 3 is obtained by dissolving dimethyl 1-(3,5-dinitrobenzyl)-1H-imidazole-4,5-dicarboxylate in about 25 ml of aqueous ammonia, and stirring at 45 ° C overnight to obtain a gray suspension. The mixture was filtered, washed with water (10 mL ⁇ 3), and dried under reduced pressure at 40 ° C to give 1.7 g of the product (1-(3,5-dinitrobenzyl)-1H-imidazole-4,5-diamide). The total yield in two steps was 75%.
  • the p-methylsulfonylbenzoic acid (12 g, 60.3 mmol, 1 eq) was dissolved in 100 ml of tetrahydrofuran, and the mixture was cooled to 0 ° C, then sodium borohydride (4.6 g, 120.6 mmol, 2.0 eq) was added, and the reaction was stirred for 1 h. Boron.
  • Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (9.2 g, 50 mmol, 1 eq), p-methylsulfonylbenzyl chloride (20.5 g, 100 mmol, 2 eq) and potassium carbonate (17.3 g, 125.0 mmol, 2.5 eq
  • the solution was dissolved in 50 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 20 h, and the reaction was monitored by TLC to the end.
  • reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL ⁇ 3), and the ethyl acetate layer was combined, washed with water (100 mL ⁇ 3) and brine (100 mL ⁇ 2), dried over anhydrous sodium sulfate. Concentrated under reduced pressure to remove the organic solvent to give the product as a colorless transparent liquid ((1-(4-(methylsulfonyl)benzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl)) .
  • the product obtained in the step 3 (1-(4-(methylsulfonyl)benzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester is dissolved in about 50 ml of aqueous ammonia, and stirred at 45 ° C overnight to obtain a white suspension. The liquid was filtered, and the filter cake was washed with water (10 mL ⁇ 3), and dried under reduced pressure at 50 ° C to give the product ((1-(4-(methylsulfonyl)benzyl)-1H-imidazole-4,5-diamide) 6.6 g. The total yield of the last two steps was 41%.
  • Step 4 Preparation of methyl 1-(4-(4-chlorophenoxy)phenyl)-1H-imidazole-4,5-dicarboxylate
  • the reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL ⁇ 3), and the ethyl acetate layer was combined, washed with water (100 mL ⁇ 3) and brine (100 mL ⁇ 2), dried over anhydrous sodium sulfate.
  • Example 13 Synergistic effect of N-benzylmidamide derivatives on colistin-sensitive Escherichia coli inhibitory activity.
  • the concentration of the N-benzylmidamide derivative was fixed at 100.0 ppm, and the colistin sulfate was subjected to gradient dilution for the purpose of testing the derivative containing 100.0 ppm of N-benzylimidamide.
  • Example 14 Synergistic effect of N-benzylmidamide derivatives on colistin-sensitive Escherichia coli inhibitory activity.
  • MIC in vitro minimum inhibitory concentration
  • the minimum inhibitory concentration of colistin sulfate to the corresponding strain in the case of containing 100.0 ppm of N-benzylimimidamide derivative respectively (the combination of N-benzylmidamide derivative and colistin sulfate as test drug)
  • concentration of the N-benzylmidamide derivative was fixed at 100.0 ppm, and the colistin sulfate was subjected to a gradient dilution for the purpose of testing the coagulizin sulfate for the corresponding strain in the case of containing 100.0 ppm of the N-benzylimimidamide derivative.
  • Minimum inhibitory concentration The results are shown in Table 3.
  • test strains were sensitive to colistin sulfate and all the test N-benzylmidamide derivatives had no inhibitory activity against the test strain.
  • the test group containing 100.0 ppm of N-benzylimimidamide derivative showed a minimum reduction in the minimum inhibitory concentration of the corresponding test strain, about 2-4 times (Table 4).
  • E. coli 3Y-9 E. coli 2S-19 E. coli 5W-7 E. coli 3B-24 Colistin sulfate 2.0 2.0 1.0 2.0 15031 >800 >800 >800 >800 15037 >800 >800 >800 >800
  • Example 15 Reversal of drug resistance of different types of colistin sulfate resistant strains with different concentrations of N-benzylmidamide derivatives (15037).
  • Tests for different types of sulfuric acid by using the test tube double dilution method for colistin sulfate and N-benzylmidamide derivatives (15037)
  • the minimum inhibitory concentration of colistin-resistant Gram-negative bacteria resistant to colistin sulfate, MIC value greater than 4.0 ppm
  • the test also contained different concentrations of N-benzylmidamide in the culture medium.
  • the minimum inhibitory concentration of colistin sulfate to the corresponding strain in the derivative (15037).
  • Example 16 Synergistic application effect of N-benzylmidamide derivative and colistin sulfate in weaned piglets.
  • Example 17 Effect of synergistic application of N-benzylmidamide derivative (15037) and colistin sulfate in weaned piglets.
  • Example 18 Effect of synergistic application of different doses of N-benzylmidamide derivative (15037) and colistin sulfate in weaned piglets.
  • Example 19 The combined use of N-benzylmidamide derivative (15037) and colistin sulfate in chicken feed.
  • Example 20 The combined use effect of N-benzylmidamide derivative (15037) and colistin sulfate in meat duck feed.
  • Each test group contained 4 parallel test groups, 50 in each parallel test group, and added Lactobacillus sulfate in each group of feeds. Or / and N-benzylmidamide derivatives (15037). Cage in the trial period, free access to food and free drinking water. The trial period was 21 days. The test results showed that the N-benzylmidamide derivative (15037) and colistin sulfate showed synergistic effects on the weight gain and feed remuneration of the test ducks (Table 10).

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Abstract

The present invention discloses the application of N-benzylimidazolecarboxamide derivatives as synergist for polymyxin antibiotics. N-benzylimidazolecarboxamide derivatives have synergistic effects for polymyxin antibiotics in the prevention and control of livestock and poultry diseases and in the improvement of the growth and production of livestock and poultry, and can be used in the field of the prevention and control of livestock and poultry diseases and the promotion of livestock and poultry growth to improve animal production performance and thus enhance the economic benefits of livestock and poultry farming. On the other hand, the combination of N-benzylimidamide derivatives and polymyxin antibiotics can reduce the dose of polymyxin antibiotics so as to reduce toxic and side effect and improve the use efficiency and use scope of polymyxin antibiotics in human pharmaceutic field.

Description

N-苄基咪酰胺衍生物作为多粘菌素类抗生素协同增效剂的应用Application of N-benzylmidamide derivatives as synergistic synergists for polymyxin antibiotics 技术领域：Technical field:
本发明属于生物医药领域，涉及N-苄基咪酰胺衍生物作为多粘菌素类抗生素协同增效剂的应用，如人用药物的制备以及作为动物防治疾病用药和饲料添加剂的应用。The invention belongs to the field of biomedicine and relates to the application of N-benzylmidamide derivatives as synergistic synergists of polymyxin antibiotics, such as preparation of human drugs and application as an animal control agent and feed additive.
背景技术：Background technique:
多粘菌素(polymyxin)是发现于多粘杆菌(Bacillus polymyxa)培养液中的抗菌性多肽，有A、B、C、D、E等五种，是结构相似，抗菌谱相互类似而范围宽广，特别对革兰氏阴性细菌有杀灭作用，临床常用的是多粘菌素B和多粘菌素E(硫酸粘杆菌素)，由于毒性较大，主要用于局部治疗敏感菌所引起的眼、耳、皮肤、粘膜感染及烧伤时铜绿假单胞菌感染以及口服用于肠道术前准备。在饲料工业中作为饲料添加剂应用，能防治畜禽疾病，还能改善畜禽新陈代谢，提高成活率和饲料转化率促进畜禽生长。Polymyxin is an antibacterial polypeptide found in Bacillus polymyxa culture medium. It has five kinds of A, B, C, D, E, etc. It is similar in structure, and the antibacterial spectrum is similar to each other and has a wide range. It has a killing effect on Gram-negative bacteria in particular. Polymyxin B and polymyxin E (colistin sulfate) are commonly used in clinical practice. Due to its high toxicity, it is mainly used for local treatment of sensitive bacteria. Infection of Pseudomonas aeruginosa in eyes, ears, skin, mucosal infections and burns and oral administration for preoperative preparation of the intestines. As a feed additive application in the feed industry, it can prevent livestock diseases, improve the metabolism of livestock and poultry, and increase the survival rate and feed conversion rate to promote the growth of livestock and poultry.
近年来，由于细菌耐药性问题，多粘菌素类抗生素在实际生产和生活中的应用大受影响。在生产实践中已有多种药物与多粘菌素类抗生素配伍使用以便增加药品的疗效，试验证明这些配伍药物与多粘菌素类抗生素联合配伍的治疗效果其实为各自对不同感染菌株的抑制效果的综合结果，而对多粘菌素类抗生素没有任何协同增效的作用。另外，这些配伍药物或为抗菌剂或为杀菌剂或为具有一定毒性的化学试剂，长期联合使用有可能会引发新型的多药耐药菌株或对环境造成严重污染，而有些配伍药物已经被欧盟禁止作为饲料添加剂使用。因此，筛选新型的低毒或无毒的非杀菌非抗菌的多粘菌素类抗生素的协同增效剂或耐药性逆转剂将是解决多粘菌素类抗生素在实际使用中遇到的困难最为有效的手段。In recent years, due to the problem of bacterial resistance, the application of polymyxins in actual production and life has been greatly affected. In the production practice, a variety of drugs have been used in combination with polymyxin antibiotics in order to increase the efficacy of the drug. The test proves that the combination of these drugs and polymyxin antibiotics is actually inhibiting the different infection strains. The combined effect of the effect, but no synergistic effect on polymyxin antibiotics. In addition, these compatible drugs may be antibacterial agents or bactericides or chemical agents with certain toxicity. Long-term combined use may cause new multi-drug resistant strains or cause serious pollution to the environment, and some compatible drugs have been adopted by the European Union. It is forbidden to be used as a feed additive. Therefore, the screening of new low-toxic or non-toxic non-bactericidal non-antibacterial polymyxin antibiotic synergists or drug resistance reversal agents will solve the difficulties encountered in the practical use of polymyxin antibiotics. The most effective means.
发明内容：Summary of the invention:
本发明的目的之一是提供一种新型、安全、有效的多粘菌素类抗生素协同增效剂。One of the objects of the present invention is to provide a novel, safe and effective synergistic synergist for polymyxins.
本发明提供的硫酸粘杆菌素协同增效剂是N-苄基咪酰胺衍生物。The colistin synergist provided by the present invention is an N-benzylmidamide derivative.
默沙东的研发人员于1958年曾经报道咪唑双酰胺及相关化合物具有抗球虫活性而被开发为抗寄生虫药(Cuckler A.C.,ect.Antiparasitic drugs.Ⅱ.Anticoccidial activity of 4,5-imidazoledicarboxamide and related compounds[J].Proceedings of the Society for Experimental Biology and Medicine,1958,98:167-70.)。广州英赛特生物技术有限公司公开的咪唑双酰胺衍生物也具有抗球虫特性而用于制备抗球虫药物应用于家鸡的饲养保健 (CN201510103326.1)。发明人首次发现N-苄基咪酰胺衍生物除了具有抗球虫的活性外还对硫酸粘杆菌素在抗动物细菌感染和促生长方面具有协同增效的作用。Merck's R&D staff reported in 1958 that imidazole bisamide and related compounds have been developed as anti-parasitic agents because of their anticoccidial activity (Cuckler AC, ect. Antiparasitic drugs. II. Anticoccidial activity of 4,5-imidazoledicarboxamide and related compounds [J]. Proceedings of the Society for Experimental Biology and Medicine, 1958, 98: 167-70.). The imidazole bisamide derivative disclosed by Guangzhou Insex Biotechnology Co., Ltd. also has anti-coccidial properties and is used for the preparation of anti-coccidial drugs for the feeding and health care of domestic chickens. (CN201510103326.1). The inventors have found for the first time that in addition to having anti-coccidial activity, the N-benzylmidamide derivative has a synergistic effect on cocciin-resistant bacterial infection and growth promotion.
因此本发明提供N-苄基咪酰胺衍生物用作多粘菌素类抗生素协同增效剂的应用，所述的N-苄基咪酰胺衍生物具有如式(Ⅰ)所示的结构：The invention therefore provides for the use of a N-benzylmidamide derivative as a synergistic synergist for a polymyxin antibiotic having a structure as shown in formula (I):
Figure PCTCN2016077918-appb-000001
Figure PCTCN2016077918-appb-000001
其中，R1和R2分别为H、卤素、NO2、S(＝O)2CH3、O-Ph、O-C1-8直链或支链烷基、C1-8直链或支链烷基或C1-8直链或支链的卤代烷基；所述的卤素为F、Cl、Br或I；所述的O-Ph中的苯基是指未被取代或被1，2，3，4个取代基取代的苯基，所述的取代基选自F、Cl、Br、I、C1-8直链或支链烷基。Wherein R 1 and R 2 are each independently H, halogen, NO 2 , S(=O) 2 CH 3 , O-Ph, OC 1-8 straight or branched alkyl, C 1-8 straight or branched Alkyl or C 1-8 straight or branched haloalkyl; said halogen is F, Cl, Br or I; said phenyl in O-Ph means unsubstituted or 1,2, 3, 4 substituent-substituted phenyl groups, said substituents being selected from the group consisting of F, Cl, Br, I, C 1-8 straight or branched alkyl groups.
在一实施方案中，所述的N-苄基咪酰胺衍生物R1优选为H，R2优选为卤素。In one embodiment, the N- benzyl-imidazole derivatives R 1 is preferably H, R 2 is preferably halogen.
在另一实施方案中，所述的N-苄基咪酰胺衍生物R1优选为H，R2优选为Cl。In another embodiment, the N- benzyl-imidazole derivatives R 1 is preferably H, R 2 is preferably Cl.
在另一实施方案中，所述的N-苄基咪酰胺衍生物优选为式(Ⅱ)所示的化合物，In another embodiment, the N-benzylmidamide derivative is preferably a compound of formula (II),
Figure PCTCN2016077918-appb-000002
Figure PCTCN2016077918-appb-000002
本发明所述的多粘菌素类抗生素选自多粘菌素A、多粘菌素B、多粘菌素C、多粘菌素D或多粘菌素E。The polymyxins of the present invention are selected from the group consisting of polymyxin A, polymyxin B, polymyxin C, polymyxin D or polymyxin E.
本发明提供的N-苄基咪酰胺衍生物在体外抑菌试验研究过程对硫酸粘杆菌素抑制细菌生长作用具有显著的协同增效效果，硫酸粘杆菌素在相应的实验中的最小抑菌浓度出现明显的下降。The N-benzylmidamide derivative provided by the invention has a remarkable synergistic effect on the inhibition of bacterial growth by colistin sulfate in an in vitro antibacterial test research process, and the minimum inhibitory concentration of colistin sulfate in the corresponding experiment There was a significant drop.
发明人对N-苄基咪酰胺衍生物的革兰氏阴性菌的最小抑制浓度测试，试验结果表明N-苄基咪酰胺衍生物对革兰氏阴性菌无抑制活性。在另外一些实施方案中，测试在培养中分别 含有相同浓度不同结构的N-苄基咪酰胺衍生物时多粘菌素类抗生素对相应敏感菌株的体外最小抑制浓度，N-苄基咪酰胺衍生物显示明显的抑菌协同增效作用。The inventors tested the minimum inhibitory concentration of Gram-negative bacteria of the N-benzylmidamide derivative, and the test results showed that the N-benzylmidamide derivative had no inhibitory activity against Gram-negative bacteria. In other embodiments, the test is separately in the culture When the N-benzylmidamide derivatives containing the same concentration of different structures were used, the minimum inhibitory concentration of polymyxin antibiotics on the corresponding sensitive strains in vitro, N-benzylmidamide derivatives showed obvious synergistic synergistic effect.
在另外一些实施方案中，测试在培养中分别含有相同浓度不同结构的N-苄基咪酰胺衍生物时多粘菌素类抗生素对相应敏感菌株的体外最小抑制浓度，优选的试验敏感菌株为对多粘菌素类抗生素敏感的大肠杆菌。在另外一些实施方案中，测试在培养中分别含有相同浓度不同结构的N-苄基咪酰胺衍生物时多粘菌素类抗生素对相应耐药菌株的体外最小抑制浓度，N-苄基咪酰胺衍生物显示明显的抑菌协同增效作用，表现为耐药逆转作用。在另外一些实施方案中，不同浓度的同种N-苄基咪酰胺衍生物对多粘菌素类抗生素耐药菌株的协同增效作用呈现剂量效应。In other embodiments, the minimum inhibitory concentration of the polymyxin antibiotic to the corresponding susceptible strain in the same concentration of the different concentration of the N-benzylmidamide derivative in the culture is tested, and the preferred test susceptible strain is Polymyxin-like antibiotic-sensitive E. coli. In other embodiments, the minimum inhibitory concentration of polymyxin antibiotics against corresponding resistant strains in the presence of N-benzylimimidamide derivatives of the same concentration and different concentrations in culture, N-benzylmidamide The derivative showed obvious antibacterial synergistic effect, which was manifested as resistance reversal. In other embodiments, synergistic effects of different concentrations of the same N-benzylmidamide derivative on polymyxin antibiotic resistant strains exhibit a dose effect.
所述的细菌为革兰氏阴性菌，包括大肠杆菌、肠杆菌属、绿脓杆菌、志贺氏菌、克雷伯氏菌、沙门氏菌、产气荚膜梭菌、金黄色葡萄球菌等。The bacteria are Gram-negative bacteria, including Escherichia coli, Enterobacter, Pseudomonas aeruginosa, Shigella, Klebsiella, Salmonella, Clostridium perfringens, Staphylococcus aureus and the like.
本发明的第二个目的是提供所述的N-苄基咪酰胺衍生物作为多粘菌素类抗生素协同增效剂的应用是用于制备动物抗细菌感染的药物的应用，如用于制备抗多粘菌素类抗生素敏感菌或耐药菌的药物。A second object of the present invention is to provide the use of the N-benzylmidamide derivative as a polymyxin antibiotic synergist for the preparation of a medicament for antibacterial infection in animals, such as for preparation A drug that is resistant to polymyxin antibiotic-sensitive or resistant bacteria.
动物常见的细菌感染症状是肠道感染导致的细菌性下痢，表现为拉稀脱水、食欲不振、消化不良、精神萎靡、体形消瘦进而死亡。细菌性下痢具有传染性，可影响同群圈养的其他畜禽的健康。多粘菌素类抗生素对动物细菌性下痢具有治疗和预防作用。The common bacterial infection symptoms of animals are bacterial diarrhea caused by intestinal infection, which is characterized by diarrhea, loss of appetite, indigestion, mental dysfunction, body weight loss and death. Bacterial diarrhea is contagious and can affect the health of other livestock in the same group. Polymyxins have therapeutic and preventive effects on bacterial sputum in animals.
在畜禽的细菌性感染腹泻的治疗，采用N-苄基咪酰胺衍生物与多粘菌素类抗生素的配伍制剂比单独使用多粘菌素类抗生素制剂表现出更好的畜禽健康状态转良率，如在断奶仔猪腹泻控制方面对多粘菌素类抗生素的协同增效作用。In the treatment of bacterial infections of diarrhea in livestock and poultry, the compatibility of N-benzylmidamide derivatives with polymyxin antibiotics shows better health status of livestock and poultry than the use of polymyxin antibiotics alone. Yield, such as the synergistic effect of polymyxin antibiotics in the control of diarrhea in weaned piglets.
在一实施方案中，本发明所述的多粘菌素类抗生素协同增效剂N-苄基咪酰胺衍生物是通过口服剂型经口服用。In one embodiment, the polymyxin antibiotic synergist N-benzylmidamide derivative of the present invention is administered orally in an oral dosage form.
所述的口服剂型除了主药成分N-苄基咪酰胺衍生物还包含有药学上可接受的载体、赋形剂、稀释剂、抗氧化剂、抗结块剂、溶媒或它们的组合。The oral dosage form comprises, in addition to the main drug component N-benzylimimidamide derivative, a pharmaceutically acceptable carrier, excipient, diluent, antioxidant, anti-caking agent, vehicle or a combination thereof.
所述的治疗用口服剂型是片剂、胶囊剂、颗粒剂、粉剂、口服液、散剂、预混剂等。The therapeutic oral dosage forms are tablets, capsules, granules, powders, oral liquids, powders, premixes and the like.
在另一实施方案中，本发明所述的多粘菌素类抗生素协同增效剂与多粘菌素类抗生素按配比制备成复方制剂。In another embodiment, the polymyxin antibiotic synergist of the present invention is prepared as a compound preparation in combination with a polymyxin antibiotic.
所述的复方制剂可经口服用，除了N-苄基咪酰胺衍生物和多粘菌素类抗生素外还含有药学上可接受的载体、赋形剂、稀释剂、抗氧化剂、抗结块剂、溶媒或它们的组合。The compound preparation can be used orally, and contains a pharmaceutically acceptable carrier, an excipient, a diluent, an antioxidant, an anti-caking agent in addition to the N-benzylmidamide derivative and the polymyxin antibiotic. , solvent or a combination thereof.
所述的复方制剂是片剂、胶囊剂、颗粒剂、粉剂、口服液、散剂、预混剂等。 The compound preparations are tablets, capsules, granules, powders, oral liquids, powders, premixes and the like.
N-苄基咪酰胺衍生物在治疗用药的使用剂量为1-200ppm，优选为10-200ppm。The N-benzylmidamide derivative is used in a therapeutic dose of from 1 to 200 ppm, preferably from 10 to 200 ppm.
N-苄基咪酰胺衍生物作为多粘菌素类抗生素协同增效剂用于抗动物细菌感染，所述的动物主要是家禽和家畜，具体包括鸡、鸭、鹅、鸽、鹌鹑、猪、牛、羊、马、兔、驴、狗、猫、狐、貂或貉。The N-benzylmidamide derivative is used as a polymyxin antibiotic synergist for anti-animal bacterial infection, and the animal is mainly poultry and livestock, specifically including chicken, duck, goose, pigeon, donkey, pig, Cow, sheep, horse, rabbit, donkey, dog, cat, fox, donkey or donkey.
本发明的第三个目的是提供所述的N-苄基咪酰胺衍生物作为多粘菌素类抗生素协同增效剂的应用是用于制备动物生长促进剂。A third object of the present invention is to provide the use of the N-benzylmidamide derivative as a polymyxin antibiotic synergist for the preparation of an animal growth promoter.
多粘菌素类抗生素在饲料加工业中作为抗生素类动物生长促进剂对改善动物的生产性能具有显著的效果，所述的N-苄基咪酰胺衍生物与多粘菌素类抗生素配伍使用具有协同增强促动物生长性能的效果。Polymyxin antibiotics have a significant effect on improving the performance of animals in the feed processing industry as an antibiotic animal growth promoter. The N-benzylmidamide derivatives are compatible with polymyxin antibiotics. Synergistically enhances the effect of promoting animal growth performance.
在一实施方案中，N-苄基咪酰胺衍生物与饲用载体、稀释剂、抗氧化剂、抗结块剂、溶媒或它们的组合制成饲料添加剂用于动物的促生长。In one embodiment, the N-benzylmidamide derivative is formulated with a feed carrier, diluent, antioxidant, anti-caking agent, vehicle, or a combination thereof to produce a feed additive for growth promotion in an animal.
在另一实施方案中，N-苄基咪酰胺衍生物和多粘菌素类抗生素与饲用载体、稀释剂、抗氧化剂、抗结块剂、溶媒或它们的组合制成复方饲料添加剂用于动物的促生长。In another embodiment, the N-benzylmidamide derivative and the polymyxin antibiotic are combined with a feeding carrier, diluent, antioxidant, anti-caking agent, vehicle, or a combination thereof to form a compound feed additive for use in The growth of animals.
所述的饲料添加剂是片剂、粉剂、散剂、颗粒剂、口服液、胶囊剂、预混剂等。The feed additive is a tablet, a powder, a powder, a granule, an oral solution, a capsule, a premix, and the like.
在一实施方案中，本发明提供的饲料添加剂以单独形式添加到动物的每日口粮中使用In one embodiment, the feed additive provided by the present invention is added to the animal's daily ration in a separate form.
在另一实施方案中，本发明提供的饲料添加剂与饲料原料、饲料添加剂、载体或稀释剂等按照一定比例制备成添加剂预混合饲料、浓缩饲料、配合饲料、精料补充饲料等饲料形式供动物食用。In another embodiment, the feed additive, the feed additive, the carrier or the diluent provided by the present invention are prepared into an additive premixed feed, concentrated feed, compound feed, concentrate supplement feed, etc. in a certain ratio for the animal. edible.
所述的饲料添加剂以N-苄基咪酰胺衍生物剂量在饲料中的使用量是1-1000ppm，优选为10-500ppm。The feed additive is used in the feed at a dose of N-benzylmidamide derivative in an amount of from 1 to 1000 ppm, preferably from 10 to 500 ppm.
N-苄基咪酰胺衍生物作为多粘菌素类抗生素协同增效剂用于动物促生长，所述的动物主要是家禽和家畜，具体包括鸡、鸭、鹅、鸽、鹌鹑、猪、牛、羊、马、兔、驴、狗、猫、狐、貂或貉。N-benzylmidamide derivatives are used as synergistic synergists for polymyxins in animals for growth. The animals are mainly poultry and livestock, including chickens, ducks, geese, pigeons, quails, pigs, and cattle. , sheep, horses, rabbits, donkeys, dogs, cats, foxes, baboons or baboons.
本发明还提供了N-苄基咪酰胺衍生物作为多粘菌素类抗生素的协同增效剂应用于制备人用抗革兰氏阴性菌感染的药物。The present invention also provides a synergistic synergist of an N-benzylmidamide derivative as a polymyxin antibiotic for the preparation of a medicament for human infection against Gram-negative bacteria.
多粘菌素类抗生素临床应用毒性大，具有神经系统毒性和瘙痒、发热和皮疹的变态反应等不良反应，限制了该药品在临床上的使用范围以及使用率。The clinical application of polymyxin antibiotics is highly toxic, and has adverse reactions such as nervous system toxicity and allergic reactions such as itching, fever and rash, which limits the clinical use and utilization rate of the drug.
N-苄基咪酰胺衍生物对多粘菌素类抗生素具有协同增效作用，与其联合应用可显著减少多粘菌素类抗生素的使用剂量从而降低毒副反应发生的概率以及强度。The N-benzylmidamide derivative has synergistic effect on polymyxin antibiotics, and the combined use thereof can significantly reduce the dosage of polymyxin antibiotics and reduce the probability and intensity of toxic side reactions.
本发明所述的N-苄基咪酰胺衍生物联合多粘菌素类抗生素在制备人用复方治疗药物中 的应用。复方治疗药物包含N-苄基咪酰胺衍生物和多粘菌素类抗生素中的一种以及其他药用辅料，可以是散剂、颗粒剂、片剂、胶囊剂、软膏剂、眼膏剂、凝胶剂、气雾剂、膜剂、口服液、滴眼剂、注射液、栓剂等。该复方治疗药物可用于人体细菌所致的胃肠道感染、败血症、尿路感染、痢疾、百日咳、呼吸道感染、胆道感染、烧伤或外伤创面感染等由革兰氏阴性菌感染引起的疾病治疗。The N-benzylmidamide derivative of the present invention is combined with polymyxin antibiotics in the preparation of a compound therapeutic drug for human use Applications. The compound therapeutic drug comprises one of N-benzylimimidamide derivative and polymyxin antibiotic and other medicinal excipients, and may be powder, granule, tablet, capsule, ointment, eye ointment, gel Agents, aerosols, films, oral liquids, eye drops, injections, suppositories, and the like. The compound therapeutic drug can be used for treating diseases caused by Gram-negative bacteria infection such as gastrointestinal infection, sepsis, urinary tract infection, dysentery, whooping cough, respiratory tract infection, biliary tract infection, burn or traumatic wound infection caused by human bacteria.
由此可见，本发明提供的N-苄基咪酰胺衍生物可作为新型有效安全的多粘菌素类抗生素配伍制剂可用于动物的疾病预防和治疗、作为协同增效剂提高所谓抗生素类动物生长促进剂的使用效率从而改善动物的生产性能提高饲养的经济效益，并能在人用药物临床使用中降低多粘菌素类抗生素的使用剂量从而减少毒副反应的发生提高药物的使用效率。It can be seen that the N-benzylmidamide derivative provided by the invention can be used as a novel effective and safe polymyxin antibiotic preparation for animal disease prevention and treatment, and as a synergistic agent to improve the growth of so-called antibiotic animals. The use efficiency of the accelerator thereby improving the production performance of the animal improves the economic benefit of the feeding, and can reduce the dosage of the polymyxin antibiotic in the clinical use of the human medicine, thereby reducing the occurrence of toxic side reactions and improving the use efficiency of the medicine.
另一方面，本发明还涉及所述N-苄基咪酰胺衍生物的制备、分离、纯化等方法和过程。In another aspect, the invention also relates to methods and processes for the preparation, isolation, purification, and the like of the N-benzylmidamide derivatives.
本发明的任一方面的任一实施方案可以与其他实施方案进行组合，只要它们之间不出现矛盾。此外，在本发明任一方面的任一实施方案中，任一技术特征可以适用于其它实施方案中的该技术特征，只要它们之间不会出现矛盾。Any of the embodiments of any of the aspects of the invention may be combined with other embodiments as long as no contradiction occurs between them. Furthermore, in any of the embodiments of any of the aspects of the present invention, any of the technical features may be applied to the technical features in other embodiments as long as there is no contradiction between them.
前面所述内容只是概述了本发明的某些方面，但不限于这些方面。The foregoing is merely an overview of some aspects of the invention, but is not limited thereto.
本发明其他方面的进一步详细描述。Further details of other aspects of the invention.
现在详细描述本发明的某些实施方案，其实例由随附的结构式和化学式加以说明。本发明的意图涵盖所有的替代、修改和等同的技术方案，它们均包括在如权利要求定义的本发明的范围内。另外，本发明的某些技术特征为清楚可见，在多个独立的实施方案中分别进行描述，但也可以在单个实施例中以组合形式提供或以任意适合的子组合形式提供。Some embodiments of the invention are now described in detail, examples of which are illustrated by the accompanying structural formulas and formulas. The invention is intended to cover all alternatives, modifications, and equivalents, which are within the scope of the invention as defined by the appended claims. In addition, some of the technical features of the present invention are clearly described in the various independent embodiments, but may be provided in combination in a single embodiment or in any suitable sub-combination.
本发明涉及的名词定义。The definition of nouns involved in the present invention.
除非另有说明，本发明涉及的名词定义具有与本领域技术人员通常理解相同的含义。Unless otherwise stated, the definition of nouns referred to in the present invention has the same meaning as commonly understood by one of ordinary skill in the art.
本发明所涉及的一些化学基团表达：“C1-4直链或支链烷基”代表含有甲基、乙基、丙基、异丙基、C4烷基直链或带支链的烷烃基；“C1-4直链或支链卤代烷基”代表甲基、乙基、丙基、异丙基、C4烷基直链或带支链的烷烃基并且烷烃基被至少一个卤素原子取代；“O-C1-4”代表烷氧基；“S(＝O)2C”代表的是烷基磺酰基。Some chemical groups involved in the present invention are expressed: "C 1-4 straight or branched alkyl" means methyl, ethyl, propyl, isopropyl, C 4 alkyl straight or branched An alkane group; "C 1-4 straight or branched haloalkyl" represents a methyl, ethyl, propyl, isopropyl, C 4 alkyl straight or branched alkane group and the alkane group is at least one halogen atom; "OC 1-4" represents an alkoxy group; "S (= O) 2 C" represents alkylsulfonyl group.
如上文式(I)所示的苯环上的取代基R1和R2通过一个单键链接到苯环的中心表示可以取代环上任一可取代位置上的氢原子。The substituents R 1 and R 2 on the benzene ring represented by the above formula (I) are linked to the center of the benzene ring by a single bond to represent a hydrogen atom at any substitutable position on the ring.
一般而言，术语“取代”表示所给结构中的一个或多个氢原子被具体取代基所取代。除非其他方面表明，一个取代的基团可以有一个或多个取代基在基团各个可取代的位置进行取代。当所给出的结构式中不只有一个位置能被选自具体基团的一个或多个取代基所取代， 那么取代基可以相同或不同地在各个位置取代。In general, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced by a particular substituent. Unless otherwise indicated, a substituted group may have one or more substituents substituted at each substitutable position of the group. When more than one position in the given formula can be substituted by one or more substituents selected from a particular group, The substituents may then be substituted at the same or different positions.
在本发明的各部分，描述了连接取代基。当该结构清楚地需要链接基团时，针对该基团所列举的马库什变量应理解为连接基团。例如，如果该结构需要连接基团并且针对该变量的马库什基团定义列举了“烷基”，则应该理解，该“烷基”代表连接的亚烷基基团。In various parts of the invention, linking substituents are described. When the structure clearly requires a linking group, the Markush variable recited for that group is understood to be a linking group. For example, if the structure requires a linking group and the Markush group definition for the variable recites "alkyl", it should be understood that the "alkyl" represents a linked alkylene group.
一般合成步骤General synthetic procedure
为了描述本发明，以下列出了实施例。但需要理解，本发明不限于这些实施例，只是提供实践本发明的方法。一般地，本发明的N-苄基咪酰胺衍生物可以通过本发明描述的方法制备得到，除非有进一步的说明，其中取代基的定义如式(I)所示。下面的反应方案和实施例用于进一步举例说明本发明的内容。For the purpose of describing the invention, the examples are set forth below. However, it is to be understood that the invention is not limited to the embodiments, but merely provides a method of practicing the invention. In general, the N-benzylmidamide derivatives of the present invention can be prepared by the methods described herein, unless otherwise stated, wherein the substituents are as defined for formula (I). The following reaction schemes and examples are provided to further illustrate the contents of the present invention.
所属领域的技术人员将认识到，本发明所描述的化学反应可以用来合适的制备许多本发明的其他化合物，且用于制备本发明的N-苄基咪酰胺衍生物的其他方法都被认为是在本发明的范围之内。例如，根据本发明那些非例正的N-苄基咪酰胺衍生物的合成可以成功的被所述领域技术人员通过修饰方法完成，如适当的保护干扰基团，通过利用其他试剂或将反应条件做一些常规修改。另外，本发明所公开的反应或已知的反应条件也公认的适用于本发明的其他化合物的制备。Those skilled in the art will recognize that the chemical reactions described herein can be used to suitably prepare a number of other compounds of the present invention, and that other methods for preparing the N-benzylmidamide derivatives of the present invention are considered It is within the scope of the invention. For example, the synthesis of non-normal N-benzylimimidamide derivatives according to the present invention can be successfully accomplished by modification methods by those skilled in the art, such as appropriate protection of interfering groups, by the use of other reagents or by reaction conditions. Make some regular modifications. Additionally, the reactions disclosed or the known reaction conditions are also recognized as suitable for the preparation of other compounds of the invention.
制备本发明的N-苄基咪酰胺衍生物的合成步骤如下面合成方案所示:The synthetic procedure for the preparation of the N-benzylmidamide derivatives of the present invention is shown in the following synthetic scheme:
Figure PCTCN2016077918-appb-000003
Figure PCTCN2016077918-appb-000003
具体实施方式：detailed description:
为了描述本发明，以下列出了实施例。但需要理解，本发明不限于这些实施例，只是提供实践本发明的方法。For the purpose of describing the invention, the examples are set forth below. However, it is to be understood that the invention is not limited to the embodiments, but merely provides a method of practicing the invention.
实施例1 1-(2-氯-6-氟苄基)-1H-咪唑-4，5-二酰胺(15031)的制备Example 1 Preparation of 1-(2-chloro-6-fluorobenzyl)-1H-imidazole-4,5-diamide (15031)
Figure PCTCN2016077918-appb-000004
Figure PCTCN2016077918-appb-000004
步骤1： 1-(2-氯-6氟苄基)-1H-咪唑-4，5-二甲酸二甲酯的制备Step 1: Preparation of dimethyl 1-(2-chloro-6fluorobenzyl)-1H-imidazole-4,5-dicarboxylate
游离的1H-咪唑-4，5-二甲酸二甲酯(9.2g，50mmol，1eq)、2-氯-6-氟苄氯(17.9g，100mmol，2eq)和碳酸钾(17.3g，125mmol，2.5eq)依次溶于70mL N,N-二甲基甲酰胺中，100℃搅拌反应15h，TLC监控反应至终点。反应液冷却至室温，加入150mL水，乙酸乙酯萃取(100mL×3)，合并乙酸乙酯层，依次水洗(100mL×3)和饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得产物为无色透明液体(1-(2-氯-6氟苄基)-1H-咪唑-4，5-二甲酸二甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (9.2 g, 50 mmol, 1 eq), 2-chloro-6-fluorobenzyl chloride (17.9 g, 100 mmol, 2 eq) and potassium carbonate (17.3 g, 125 mmol, 2.5 eq) was dissolved in 70 mL of N,N-dimethylformamide, and the reaction was stirred at 100 ° C for 15 h, and the reaction was monitored by TLC to the end. The reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL×3), and the ethyl acetate layer was combined, washed with water (100 mL×3) and brine (100 mL×2), dried over anhydrous sodium sulfate. The organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(2-chloro-6fluorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester), which was directly used for the next reaction.
步骤2： 1-(2-氯-6-氟苄基)-1H-咪唑-4，5-二酰胺的制备Step 2: Preparation of 1-(2-chloro-6-fluorobenzyl)-1H-imidazole-4,5-diamide
步骤1所得的产物1-(2-氯-6氟苄基)-1H-咪唑-4，5-二甲酸二甲酯溶于约50ml氨水中，45℃搅拌反应过夜，得白色混悬液，过滤，滤饼用水洗涤(20mL×3)，55℃减压干燥，得产物(1-(2-氯-6-氟苄基)-1H-咪唑-4，5-二酰胺)11.8g。总产率79.8％。The product obtained in the first step, 1-(2-chloro-6fluorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester, is dissolved in about 50 ml of aqueous ammonia, and stirred at 45 ° C overnight to obtain a white suspension. After filtration, the filter cake was washed with water (20 mL × 3), and dried under reduced pressure at 55 ° C to give 11.1 g of the product (1-(2-chloro-6-fluorobenzyl)-1H-imidazole-4,5-diamide). The total yield was 79.8%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.563(1H，s)，7.954(1H，s)，7.794(1H，s)，7.659(1H，s)，7.579(1H，s)，7.424-7.468(1H，m),7.370-7.386(1H，d)，7.229-7.268(1H，m)，5.826(2H，s)。 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 10.563 (1H, s), 7.954 (1H, s), 7.794 (1H, s), 7.659 (1H, s), 7.579 (1H, s) , 7.424-7.468 (1H, m), 7.370-7.386 (1H, d), 7.229-7.268 (1H, m), 5.826 (2H, s).
实施例2 1-(4-氯苄基)-1H-咪唑-4，5-二酰胺(15037)的制备Example 2 Preparation of 1-(4-chlorobenzyl)-1H-imidazole-4,5-diamide (15037)
Figure PCTCN2016077918-appb-000005
Figure PCTCN2016077918-appb-000005
步骤1： 1-(4-氯苄基)-1H-咪唑-4，5-二甲酸二甲酯的制备Step 1: Preparation of dimethyl 1-(4-chlorobenzyl)-1H-imidazole-4,5-dicarboxylate
游离的1H-咪唑-4，5-二甲酸二甲酯(7.6g，41mmol，1eq)、对氯苄氯(13.2g，82mmol，2eq)和碳酸钾(14.2g，103mmol，2.5eq)依次溶于50mL N,N-二甲基甲酰胺中，90℃搅拌反应16h，TLC监控反应至终点。反应液冷却至室温，加入150mL水，乙酸乙酯萃取(100mL×3)，合并乙酸乙酯层，依次水洗(100mL×3)和饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得产物为无色透明液体(1-(4-氯苄基)-1H-咪唑-4，5-二甲酸二甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (7.6 g, 41 mmol, 1 eq), p-chlorobenzyl chloride (13.2 g, 82 mmol, 2 eq) and potassium carbonate (14.2 g, 103 mmol, 2.5 eq) The reaction was stirred at 90 ° C for 16 h in 50 mL of N,N-dimethylformamide and the reaction was monitored by TLC. The reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL×3), and the ethyl acetate layer was combined, washed with water (100 mL×3) and brine (100 mL×2), dried over anhydrous sodium sulfate. The organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(4-chlorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
步骤2： 1-(4-氯苄基)-1H-咪唑-4，5-二酰胺的制备Step 2: Preparation of 1-(4-chlorobenzyl)-1H-imidazole-4,5-diamide
步骤1所得的产物1-(4-氯苄基)-1H-咪唑-4，5-二甲酸二甲酯溶于约50ml氨水中，45℃搅拌反应过夜，得白色混悬液，过滤，滤饼用水洗涤(20mL×3)，55℃减压干燥，得产物(1-(4-氯苄基)-1H-咪唑-4，5-二酰胺)5.0g。总产率44％。 The product obtained in the first step, 1-(4-chlorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester, is dissolved in about 50 ml of aqueous ammonia, and stirred at 45 ° C overnight to obtain a white suspension, which is filtered and filtered. The cake was washed with water (20 mL × 3), and dried under reduced pressure at 55 ° C to give the product (1-(4-chlorobenzyl)-1H-imidazole-4,5-diamide) 5.0 g. The total yield was 44%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.931(1H，s)，7.6(1H，s)，7.505(1H，s)，7.301-7.318(2H，d)，7.135-7.152(2H，d)，5.708(2H，s)，5.578-5.603(2H，s)。 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 10.931 (1H, s), 7.6 (1H, s), 7.505 (1H, s), 7.301-7.318 (2H, d), 7.135-7.152 ( 2H, d), 5.708 (2H, s), 5.558-5.603 (2H, s).
实施例3 1-(3-氯苄基)-1H-咪唑-4，5-二酰胺(15041)的制备Example 3 Preparation of 1-(3-chlorobenzyl)-1H-imidazole-4,5-diamide (15041)
Figure PCTCN2016077918-appb-000006
Figure PCTCN2016077918-appb-000006
步骤1： 1-(3-氯苄基)-1H-咪唑-4，5-二甲酸二甲酯的制备Step 1: Preparation of dimethyl 1-(3-chlorobenzyl)-1H-imidazole-4,5-dicarboxylate
游离的1H-咪唑-4，5-二甲酸二甲酯(5.0g，27mmol，1eq)、3-氯苄溴(11.5g，56mmol，2eq)和碳酸钾(9.3g，67.5mmol，2.5eq)依次溶于50mL N,N-二甲基甲酰胺中，90℃搅拌反应10h，TLC监控反应至终点。反应液冷却至室温，加入150mL水，乙酸乙酯萃取(100mL×3)，合并乙酸乙酯层，依次水洗(100mL×3)和饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得产物为无色透明液体(1-(3-氯苄基)-1H-咪唑-4，5-二甲酸二甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (5.0 g, 27 mmol, 1 eq), 3-chlorobenzyl bromide (11.5 g, 56 mmol, 2 eq) and potassium carbonate (9.3 g, 67.5 mmol, 2.5 eq) The solution was dissolved in 50 mL of N,N-dimethylformamide, and the reaction was stirred at 90 ° C for 10 h, and the reaction was monitored by TLC to the end. The reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL×3), and the ethyl acetate layer was combined, washed with water (100 mL×3) and brine (100 mL×2), dried over anhydrous sodium sulfate. The organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(3-chlorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
步骤2： 1-(3-氯苄基)-1H-咪唑-4，5-二酰胺的制备Step 2: Preparation of 1-(3-chlorobenzyl)-1H-imidazole-4,5-diamide
步骤1所得的产物1-(3-氯苄基)-1H-咪唑-4，5-二甲酸二甲酯溶于约50ml氨水中，45℃搅拌反应过夜，得白色混悬液，过滤，滤饼用水洗涤(20mL×3)，55℃减压干燥，得产物(1-(3-氯苄基)-1H-咪唑-4，5-二酰胺)5.3克。总产率71％。The product obtained in the first step, 1-(3-chlorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester, is dissolved in about 50 ml of aqueous ammonia, and stirred at 45 ° C overnight to obtain a white suspension, which is filtered and filtered. The cake was washed with water (20 mL × 3), and dried under reduced pressure at 55 ° C to give 5.3 g of product (1-(3-chlorobenzyl)-1H-imidazole-4,5-diamide). The total yield was 71%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.641(1H，s)，8.138(1H，s)，8.009(1H，s)，7.821(1H，s)，7.556(1H，s)，7.331-7.369(2H，m)，7.240(1H，s)，7.094-7.108(1H，d)，5.713(2H，s)。 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 10.641 (1H, s), 8.138 (1H, s), 8.009 (1H, s), 7.821 (1H, s), 7.556 (1H, s) , 7.331-7.369 (2H, m), 7.240 (1H, s), 7.094-7.108 (1H, d), 5.713 (2H, s).
实施例4 1-(4-氟苄基)-1H-咪唑-4，5-二酰胺(15042)的制备Example 4 Preparation of 1-(4-fluorobenzyl)-1H-imidazole-4,5-diamide (15042)
Figure PCTCN2016077918-appb-000007
Figure PCTCN2016077918-appb-000007
步骤1： 1-(4-氟苄基)-1H-咪唑-4，5-二甲酸二甲酯的制备Step 1: Preparation of dimethyl 1-(4-fluorobenzyl)-1H-imidazole-4,5-dicarboxylate
游离的1H-咪唑-4，5-二甲酸二甲酯(8.37g，45.5mmol，1eq)、对氟苄氯(13.1g，91mmol，2eq)和碳酸钾(18.5g，133.8mmol，2.5eq)依次溶于70mL N,N-二甲基甲酰胺中，90℃搅拌反应10h，TLC监控反应至终点。反应液冷却至室温，加入150mL水，乙酸乙酯萃取(100mL ×3)，合并乙酸乙酯层，依次水洗(100mL×3)和饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得产物为无色透明液体(1-(4-氟苄基)-1H-咪唑-4，5-二甲酸二甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (8.37 g, 45.5 mmol, 1 eq), p-fluorobenzyl chloride (13.1 g, 91 mmol, 2 eq) and potassium carbonate (18.5 g, 133.8 mmol, 2.5 eq) The solution was dissolved in 70 mL of N,N-dimethylformamide, and the reaction was stirred at 90 ° C for 10 h, and the reaction was monitored by TLC to the end. The reaction solution was cooled to room temperature, added with 150 mL of water and ethyl acetate (100 mL) ×3), the ethyl acetate layer was combined, washed with water (100 mL×3) and brine (100 mL×2), dried over anhydrous sodium sulfate, and evaporated. (4-Fluorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester) was used directly in the next reaction.
步骤2： 1-(4-氟苄基)-1H-咪唑-4，5-二酰胺的制备Step 2: Preparation of 1-(4-fluorobenzyl)-1H-imidazole-4,5-diamide
步骤1所得的产物1-(4-氟苄基)-1H-咪唑-4，5-二甲酸二甲酯溶于约50ml氨水中，45℃搅拌反应过夜，得白色混悬液，过滤，滤饼用水洗涤(20mL×3)，55℃减压干燥，得产物(1-(4-氟苄基)-1H-咪唑-4，5-二酰胺)2.9克。总产率24.4％。The product obtained in the first step, 1-(4-fluorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester is dissolved in about 50 ml of aqueous ammonia, and stirred at 45 ° C overnight to obtain a white suspension, which is filtered and filtered. The cake was washed with water (20 mL × 3), and dried under reduced pressure at 55 ° C to give the product (1-(4-fluorobenzyl)-1H-imidazole-4,5-diamide) 2.9 g. The total yield was 24.4%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.622(1H，s)，8.110(1H，s)，7.985(1H，s)，7.795(1H，s)，7.534(1H，s)，7.229-7.258(2H，m)，7.127-7.162(2H，m)，5.696(2H，s)。 1 H NMR (500 MHz, DMSO-d 6 ): δ (ppm) 10.622 (1H, s), 8.10 (1H, s), 7.985 (1H, s), 7.795 (1H, s), 7.534 (1H, s) , 7.229-7.258 (2H, m), 7.127-7.162 (2H, m), 5.696 (2H, s).
实施例5 1-(4-硝基苄基)-1H-咪唑-4，5-二酰胺(15045)的制备Example 5 Preparation of 1-(4-nitrobenzyl)-1H-imidazole-4,5-diamide (15045)
Figure PCTCN2016077918-appb-000008
Figure PCTCN2016077918-appb-000008
步骤1： 1-(4-硝基苄基)-1H-咪唑-4，5-二甲酸二甲酯的制备Step 1: Preparation of dimethyl 1-(4-nitrobenzyl)-1H-imidazole-4,5-dicarboxylate
游离的1H-咪唑-4，5-二甲酸二甲酯(6.0g，32.6mmol，1eq)、对硝基苄氯(11.2g，65.2mmol，2eq)和碳酸钾(11.3g，81.5mmol，2.5eq)依次溶于50mL N,N-二甲基甲酰胺中，120℃搅拌反应15h，TLC监控反应至终点。反应液冷却至室温，加入150mL水，乙酸乙酯萃取(100mL×3)，合并乙酸乙酯层，依次水洗(100mL×3)和饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得产物为无色透明液体(1-(4-硝基苄基)-1H-咪唑-4，5-二甲酸二甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (6.0 g, 32.6 mmol, 1 eq), p-nitrobenzyl chloride (11.2 g, 65.2 mmol, 2 eq) and potassium carbonate (11.3 g, 81.5 mmol, 2.5 Eq) was dissolved in 50 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 15 h, and the reaction was monitored by TLC to the end. The reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL×3), and the ethyl acetate layer was combined, washed with water (100 mL×3) and brine (100 mL×2), dried over anhydrous sodium sulfate. The organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(4-nitrobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
步骤2： 1-(4-硝基苄基)-1H-咪唑-4，5-二酰胺的制备Step 2: Preparation of 1-(4-nitrobenzyl)-1H-imidazole-4,5-diamide
步骤1所得的产物1-(4-硝基苄基)-1H-咪唑-4，5-二甲酸二甲酯溶于约50ml氨水中，45℃搅拌反应过夜，得白色混悬液，过滤，滤饼用水洗涤(20mL×3)，55℃减压干燥，得产物(1-(4-硝基苄基)-1H-咪唑-4，5-二酰胺)4.8g。总产率50.5％。The product obtained in the first step is obtained by dissolving the product of 1-(4-nitrobenzyl)-1H-imidazole-4,5-dicarboxylate in about 50 ml of aqueous ammonia, and stirring at 45 ° C overnight to obtain a white suspension, which is filtered. The filter cake was washed with water (20 mL × 3), and dried under reduced pressure at 55 ° C to give 4.8 g of the product (1-(4-nitrobenzyl)-1H-imidazole-4,5-diamide). The total yield was 50.5%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.664(1H，s)，8.161-8.195(3H，m)，8.041(1H，s)，7.847(1H，s)，7.526(1H，s)，7.329-7.347(2H，d)，5.839(2H，s)。 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 10.664 (1H, s), 8.161-8.195 (3H, m), 8.041 (1H, s), 7.847 (1H, s), 7.526 (1H, s), 7.329-7.347 (2H, d), 5.839 (2H, s).
实施例6 1-(4-甲基苄基)-1H-咪唑-4，5-二酰胺(15046)的制备 Example 6 Preparation of 1-(4-methylbenzyl)-1H-imidazole-4,5-diamide (15046)
Figure PCTCN2016077918-appb-000009
Figure PCTCN2016077918-appb-000009
步骤1： 1-(4-甲基苄基)-1H-咪唑-4，5-二甲酸二甲酯的制备Step 1: Preparation of dimethyl 1-(4-methylbenzyl)-1H-imidazole-4,5-dicarboxylate
游离的1H-咪唑-4，5-二甲酸二甲酯(7.0g，38.0mmol，1eq)、对甲基苄氯(10.7g，76.0mmol，2eq)和碳酸钾(13.1g，95.0mmol，2.5eq)依次溶于50mL N,N-二甲基甲酰胺中，120℃搅拌反应15h，TLC监控反应至终点。反应液冷却至室温，加入150mL水，乙酸乙酯萃取(100mL×3)，合并乙酸乙酯层，依次水洗(100mL×3)和饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得产物为无色透明液体(1-(4-甲基苄基)-1H-咪唑-4，5-二甲酸二甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (7.0 g, 38.0 mmol, 1 eq), p-methylbenzyl chloride (10.7 g, 76.0 mmol, 2 eq) and potassium carbonate (13.1 g, 95.0 mmol, 2.5 Eq) was dissolved in 50 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 15 h, and the reaction was monitored by TLC to the end. The reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL×3), and the ethyl acetate layer was combined, washed with water (100 mL×3) and brine (100 mL×2), dried over anhydrous sodium sulfate. The organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(4-methylbenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
步骤2： 1-(4-甲基苄基)-1H-咪唑-4，5-二酰胺的制备Step 2: Preparation of 1-(4-methylbenzyl)-1H-imidazole-4,5-diamide
步骤1所得的产物1-(4-甲基苄基)-1H-咪唑-4，5-二甲酸二甲酯溶于约50ml氨水中，45℃搅拌反应过夜，得白色混悬液，过滤，滤饼用水洗涤(20mL×3)，55℃减压干燥，得产物(1-(4-甲基苄基)-1H-咪唑-4，5-二酰胺)7.8克。总产率79％。The product obtained in the first step is obtained by dissolving the product of 1-(4-methylbenzyl)-1H-imidazole-4,5-dicarboxylate in about 50 ml of aqueous ammonia, and stirring at 45 ° C overnight to obtain a white suspension, which is filtered. The filter cake was washed with water (20 mL × 3), and dried under reduced pressure at 55 ° C to give y. The total yield was 79%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.583(1H，s)，8.069(1H，s)，7.968(1H，s)，7.776(1H，s)，7.503(1H，s)，7.126-7.110(2H，d)，7.074-7.058(2H，d),5.671(2H，s)，2.254(3H，s)。 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 10.583 (1H, s), 8.069 (1H, s), 7.968 (1H, s), 7.776 (1H, s), 7.503 (1H, s) , 7.126-7.110 (2H, d), 7.074-7.058 (2H, d), 5.671 (2H, s), 2.254 (3H, s).
实施例7 1-(4-三氟甲基苄基)-1H-咪唑-4，5-二酰胺(15051)的制备Example 7 Preparation of 1-(4-Trifluoromethylbenzyl)-1H-imidazole-4,5-diamide (15051)
Figure PCTCN2016077918-appb-000010
Figure PCTCN2016077918-appb-000010
步骤1： 1-(4-三氟甲基苄基)-1H-咪唑-4，5-二甲酸二甲酯的制备Step 1: Preparation of dimethyl 1-(4-trifluoromethylbenzyl)-1H-imidazole-4,5-dicarboxylate
游离的1H-咪唑-4，5-二甲酸二甲酯(7.0g，38.0mmol，1eq)、对三氟甲基苄溴(18.2g，76.0mmol，2eq)和碳酸钾(13.1g，95.0mmol，2.5eq)依次溶于50mL N,N-二甲基甲酰胺中，120℃搅拌反应15h，TLC监控反应至终点。反应液冷却至室温，加入150mL水，乙酸乙酯萃取(100mL×3)，合并乙酸乙酯层，依次水洗(100mL×3)和饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得产物为无色透明液体(1-(4-三氟甲基苄基)-1H-咪唑-4，5-二甲酸二甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (7.0 g, 38.0 mmol, 1 eq), p-trifluoromethylbenzyl bromide (18.2 g, 76.0 mmol, 2 eq) and potassium carbonate (13.1 g, 95.0 mmol) , 2.5 eq) was sequentially dissolved in 50 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 15 h, and the reaction was monitored by TLC to the end. The reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL×3), and the ethyl acetate layer was combined, washed with water (100 mL×3) and brine (100 mL×2), dried over anhydrous sodium sulfate. The organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(4-trifluoromethylbenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
步骤2: 1-(4-三氟甲基苄基)-1H-咪唑-4，5-二酰胺的制备 Step 2: Preparation of 1-(4-trifluoromethylbenzyl)-1H-imidazole-4,5-diamide
步骤1所得的产物1-(4-三氟甲基苄基)-1H-咪唑-4，5-二甲酸二甲酯溶于约50ml氨水中，45℃搅拌反应过夜，得白色混悬液，过滤，滤饼用水洗涤(20mL×3)，55℃减压干燥，得产物(1-(4-三氟甲基苄基)-1H-咪唑-4，5-二酰胺)8.73克。总产率73％。The product obtained in the first step, 1-(4-trifluoromethylbenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester, is dissolved in about 50 ml of aqueous ammonia, and stirred at 45 ° C overnight to obtain a white suspension. After filtration, the filter cake was washed with water (20 mL × 3), and dried under reduced pressure at 55 ° C to give the product (1-(4-trifluoromethylbenzyl)-1H-imidazole-4,5-diamide) 8.73 g. The total yield was 73%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.651(1H，s)，8.149(1H，s)，8.026(1H，s)，7.833(1H，s)，7.697-7.681(2H，d)，7.524(1H，s)，7.319-7.303(2H，d),5.808(2H，s)。 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 10.651 (1H, s), 8.149 (1H, s), 8.026 (1H, s), 7.833 (1H, s), 7.697-7.681 (2H, d), 7.524 (1H, s), 7.319-7.303 (2H, d), 5.808 (2H, s).
实施例8 1-(4-甲氧基苄基)-1H-咪唑-4，5-二酰胺(15053)的制备Example 8 Preparation of 1-(4-methoxybenzyl)-1H-imidazole-4,5-diamide (15053)
Figure PCTCN2016077918-appb-000011
Figure PCTCN2016077918-appb-000011
步骤1： 1-(4-甲氧基苄基)-1H-咪唑-4，5-二甲酸二甲酯的制备Step 1: Preparation of dimethyl 1-(4-methoxybenzyl)-1H-imidazole-4,5-dicarboxylate
游离的1H-咪唑-4，5-二甲酸二甲酯(9.2g，50mmol，1eq)、对甲氧基苄氯(15.7g，100.0mmol，2eq)和碳酸钾(17.3g，125.0mmol，2.5eq)依次溶于70mL N,N-二甲基甲酰胺中，120℃搅拌反应15h，TLC监控反应至终点。反应液冷却至室温，加入150mL水，乙酸乙酯萃取(100mL×3)，合并乙酸乙酯层，依次水洗(100mL×3)和饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得产物为无色透明液体(1-(4-甲氧基苄基)-1H-咪唑-4，5-二甲酸二甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (9.2 g, 50 mmol, 1 eq), p-methoxybenzyl chloride (15.7 g, 100.0 mmol, 2 eq) and potassium carbonate (17.3 g, 125.0 mmol, 2.5 Eq) was dissolved in 70 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 15 h, and the reaction was monitored by TLC to the end. The reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL×3), and the ethyl acetate layer was combined, washed with water (100 mL×3) and brine (100 mL×2), dried over anhydrous sodium sulfate. The organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(4-methoxybenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
步骤2： 1-(4-甲氧基苄基)-1H-咪唑-4，5-二酰胺的制备Step 2: Preparation of 1-(4-methoxybenzyl)-1H-imidazole-4,5-diamide
步骤1所得的产物1-(4-甲氧基苄基)-1H-咪唑-4，5-二甲酸二甲酯溶于约50ml氨水中，45℃搅拌反应过夜，得白色混悬液，过滤，滤饼用水洗涤(20mL×3)，55℃减压干燥，得产物(1-(4-甲氧基苄基)-1H-咪唑-4，5-二酰胺)8.55克。总产率62％。The product obtained in the first step is obtained by dissolving the product of 1-(4-methoxybenzyl)-1H-imidazole-4,5-dicarboxylic acid in about 50 ml of aqueous ammonia, and stirring at 45 ° C overnight to obtain a white suspension. The filter cake was washed with water (20 mL × 3), and dried under reduced pressure at 55 ° C to give the product (1-(4-methoxybenzyl)-1H-imidazole-4,5-diamide) 8.55 g. The total yield was 62%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.587(1H，s)，8.069(1H，s)，7.957(1H，s)，7.766(1H，s)，7.519(1H，s)，7.180-7.163(2H，d)，6.881-6.864(2H，d),5.640(2H，s)。 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 10.587 (1H, s), 8.069 (1H, s), 7.957 (1H, s), 7.766 (1H, s), 7.519 (1H, s) , 7.180-7.163 (2H, d), 6.881-6.864 (2H, d), 5.640 (2H, s).
实施例9 1-(3，4-二氯苄基)-1H-咪唑-4，5-二酰胺(15054)的制备Example 9 Preparation of 1-(3,4-Dichlorobenzyl)-1H-imidazole-4,5-diamide (15054)
Figure PCTCN2016077918-appb-000012
Figure PCTCN2016077918-appb-000012
步骤1： 1-(3，4-二氯苄基)-1H-咪唑-4，5-二甲酸二甲酯的制备Step 1: Preparation of dimethyl 1-(3,4-dichlorobenzyl)-1H-imidazole-4,5-dicarboxylate
游离的1H-咪唑-4，5-二甲酸二甲酯(9.2g，50mmol，1eq)、3，4-二氯苄溴(24.0g， 100.0mmol，2eq)和碳酸钾(17.3g，125.0mmol，2.5eq)依次溶于70mL N,N-二甲基甲酰胺中，110℃搅拌反应14h，TLC监控反应至终点。反应液冷却至室温，加入150mL水，乙酸乙酯萃取(100mL×3)，合并乙酸乙酯层，依次水洗(100mL×3)和饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得产物为无色透明液体(1-(3，4-二氯苄基)-1H-咪唑-4，5-二甲酸二甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (9.2 g, 50 mmol, 1 eq), 3,4-dichlorobenzyl bromide (24.0 g, 100.0 mmol, 2 eq) and potassium carbonate (17.3 g, 125.0 mmol, 2.5 eq) were sequentially dissolved in 70 mL of N,N-dimethylformamide, and the reaction was stirred at 110 ° C for 14 h, and the reaction was monitored by TLC to the end. The reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL×3), and the ethyl acetate layer was combined, washed with water (100 mL×3) and brine (100 mL×2), dried over anhydrous sodium sulfate. The organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(3,4-dichlorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
步骤2： 1-(3，4-二氯苄基)-1H-咪唑-4，5-二酰胺的制备Step 2: Preparation of 1-(3,4-dichlorobenzyl)-1H-imidazole-4,5-diamide
步骤1所得的产物1-(3，4-二氯苄基)-1H-咪唑-4，5-二甲酸二甲酯溶于约50ml氨水中，45℃搅拌反应过夜，得白色混悬液，过滤，滤饼用水洗涤(20mL×3)，55℃减压干燥，得产物(1-(3，4-二氯苄基)-1H-咪唑-4，5-二酰胺)11.8克。总产率75％。The product obtained in the first step, 1-(3,4-dichlorobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester, is dissolved in about 50 ml of aqueous ammonia, and stirred at 45 ° C overnight to obtain a white suspension. After filtration, the filter cake was washed with water (20 mL × 3), and dried under reduced pressure at 55 ° C to give 11.1 g of the product (1-(3,4-dichlorobenzyl)-1H-imidazole-4,5-diamide). The total yield is 75%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.653(1H，s)，8.146(1H，s)，8.014(1H，s)，7.827(1H，s)，7.595-7.569(2H，m)，7.473(1H，s)，7.131-7.111(1H，d),5.689(2H，s)。 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 10.653 (1H, s), 8.146 (1H, s), 8.014 (1H, s), 7.827 (1H, s), 7.595-7.569 (2H, m), 7.473 (1H, s), 7.131-7.111 (1H, d), 5.689 (2H, s).
实施例10 1-(3，5-二硝基苄基)-1H-咪唑-4，5-二酰胺(15056)的制备Example 10 Preparation of 1-(3,5-dinitrobenzyl)-1H-imidazole-4,5-diamide (15056)
Figure PCTCN2016077918-appb-000013
Figure PCTCN2016077918-appb-000013
步骤1： 3，5-二硝基苄醇的制备Step 1: Preparation of 3,5-dinitrobenzyl alcohol
3，5-二硝基苯甲酸(12g，56.6mmol，1eq)溶于100ml四氢呋喃中，降温至0℃后加入硼氢化钠(4.3g，113.2mmol，2.0eq)，搅拌反应1h，缓慢滴加三氟化硼.乙醚(40.2g，283.0mmol，5eq)，逐渐升温至室温，搅拌反应过夜得浓稠反应物，缓慢倒入冷稀盐酸水溶液中，乙酸乙酯萃取(150mL×3)，合并有机层、饱和食盐水洗涤、无水硫酸钠干燥、减压浓缩除得浸膏状粗产物，经柱层析(PE/EA＝1/1)分离纯化得产物(3，5-二硝基苄醇)9.4g，产率84.1％。3,5-Dinitrobenzoic acid (12 g, 56.6 mmol, 1 eq) was dissolved in 100 ml of tetrahydrofuran. After cooling to 0 ° C, sodium borohydride (4.3 g, 113.2 mmol, 2.0 eq) was added, and the reaction was stirred for 1 h, slowly added dropwise. Boron trifluoride. Diethyl ether (40.2 g, 283.0 mmol, 5 eq), gradually warmed to room temperature, stirred overnight to give a thick reaction mixture, slowly poured into cold dilute aqueous hydrochloric acid, ethyl acetate (150 mL × 3), combined The organic layer is washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness, and then evaporated to give the crude product as a crude product, which is purified by column chromatography (PE/EA=1/1) (3,5-dinitro) Benzyl alcohol) 9.4 g, yield 84.1%.
1H NMR(500MHz，DMSO-d6):δ(ppm)8.718(1H，s)，8.579(2H，s)，5.796(1H，m)，4.763(2H，d)。 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 8.718 (1H, s), 8.579 (2H, s), 5.796 (1H, m), 4.763 (2H, d).
步骤2： 3，5-二硝基苄氯的制备Step 2: Preparation of 3,5-dinitrobenzyl chloride
3，5-二硝基苄醇(2.4g,12.12mmol,1eq)溶于干燥50mL二氯甲烷中，降温至0℃，加入三乙胺(4.9g,48.2mmol,4eq),缓慢滴加氯化亚砜(2.9g,24.2mmol,2eq)，滴毕，逐渐升温至回流，反应3h，停止加热冷却至室温，滴加50mL水淬灭反应，分层。有机层用无水硫酸钠干燥、减压浓缩除去有机溶剂得粗产物，经硅胶柱层析(PE/EA＝4/3)得产物(3，5- 二硝基苄氯)2.5g,产率95.3％。3,5-Dinitrobenzyl alcohol (2.4 g, 12.12 mmol, 1 eq) was dissolved in dry 50 mL dichloromethane, cooled to 0 ° C, triethylamine (4.9 g, 48.2 mmol, 4 eq) Sulfoxide (2.9 g, 24.2 mmol, 2 eq) was added dropwise, gradually warmed to reflux, reacted for 3 h, then the mixture was cooled and cooled to room temperature, and 50 mL of water was added dropwise to quench the reaction and the layers were separated. The organic layer was dried over anhydrous sodium sulfate and evaporated, evaporated, evaporated, evaporated. Dinitrobenzyl chloride) 2.5 g, yield 95.3%.
步骤3： 1-(3，5-二硝基苄基)-1H-咪唑-4，5-二甲酸二甲酯的制备Step 3: Preparation of dimethyl 1-(3,5-dinitrobenzyl)-1H-imidazole-4,5-dicarboxylate
游离的1H-咪唑-4，5-二甲酸二甲酯(1.28g，6.94mmol，1eq)、3，5-二硝基苄氯(3.0g，13.9mmol，2eq)和碳酸钾(2.4g，17.4mmol，2.5eq)依次溶于5mL N,N-二甲基甲酰胺中，120℃搅拌反应17h，TLC监控反应至终点。反应液冷却至室温，加入15mL水，乙酸乙酯萃取(10mL×3)，合并乙酸乙酯层，依次水洗(10mL×3)和饱和食盐水洗涤(10mL×2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得产物为无色透明液体(1-(3，5-二硝基苄基)-1H-咪唑-4，5-二甲酸二甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (1.28 g, 6.94 mmol, 1 eq), 3,5-dinitrobenzyl chloride (3.0 g, 13.9 mmol, 2 eq) and potassium carbonate (2.4 g, 17.4 mmol, 2.5 eq) was dissolved in 5 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 17 h, and the reaction was monitored by TLC to the end. The reaction mixture was cooled to room temperature, and 15 mL of water was added, and ethyl acetate (10 mL×3) was added, and the ethyl acetate layer was combined, washed with water (10 mL×3) and brine (10 mL×2) and dried over anhydrous sodium sulfate. The organic solvent was concentrated under reduced pressure to give the product as a colorless transparent liquid (1-(3,5-dinitrobenzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester).
步骤4： 1-(3，5-二硝基苄基)-1H-咪唑-4，5-二酰胺的制备Step 4: Preparation of 1-(3,5-dinitrobenzyl)-1H-imidazole-4,5-diamide
步骤3所得的产物1-(3，5-二硝基苄基)-1H-咪唑-4，5-二甲酸二甲酯溶于约25ml氨水中，45℃搅拌反应过夜，得灰色混悬液，过滤，滤饼用水洗涤(10mL×3)，40℃减压干燥，得产物(1-(3，5-二硝基苄基)-1H-咪唑-4，5-二酰胺)1.7克。两步总产率75％。The product obtained in the step 3 is obtained by dissolving dimethyl 1-(3,5-dinitrobenzyl)-1H-imidazole-4,5-dicarboxylate in about 25 ml of aqueous ammonia, and stirring at 45 ° C overnight to obtain a gray suspension. The mixture was filtered, washed with water (10 mL × 3), and dried under reduced pressure at 40 ° C to give 1.7 g of the product (1-(3,5-dinitrobenzyl)-1H-imidazole-4,5-diamide). The total yield in two steps was 75%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.727(1H，s)，8.748(1H，s)，8.517(2H，s)，8.287(1H，s)，8.062(1H，s)，7.871(1H，s)，7.665(1H，s),5.908(2H，s). 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 10.727 (1H, s), 8.748 (1H, s), 8.517 (2H, s), 8.287 (1H, s), 8.062 (1H, s) , 7.871 (1H, s), 7.665 (1H, s), 5.908 (2H, s).
实施例11(1-(4-(甲磺酰基)苄基)-1H-咪唑-4，5-二酰胺(15150)的制备Preparation of Example 11 (1-(4-(Methanesulfonyl)benzyl)-1H-imidazole-4,5-diamide (15150)
Figure PCTCN2016077918-appb-000014
Figure PCTCN2016077918-appb-000014
步骤1： 4-甲砜基苄醇的制备Step 1: Preparation of 4-methylsulfonylbenzyl alcohol
对甲砜基苯甲酸(12g，60.3mmol，1eq)溶于100ml四氢呋喃中，降温至0℃后加入硼氢化钠(4.6g，120.6mmol，2.0eq)，搅拌反应1h，缓慢滴加三氟化硼.乙醚(42.8g，301.5mmol，5eq)，逐渐升温至室温，搅拌反应过夜得浓稠反应物，缓慢倒入冰水中，乙酸乙酯萃取(150mL×3)，合并有机层、饱和食盐水洗涤、无水硫酸钠干燥、减压浓缩除得浸膏状粗产物，经柱层析(PE/EA＝1/1)分离纯化得产物(4-甲砜基苄醇)9.2g，产率81.9％。The p-methylsulfonylbenzoic acid (12 g, 60.3 mmol, 1 eq) was dissolved in 100 ml of tetrahydrofuran, and the mixture was cooled to 0 ° C, then sodium borohydride (4.6 g, 120.6 mmol, 2.0 eq) was added, and the reaction was stirred for 1 h. Boron. Diethyl ether (42.8 g, 301.5 mmol, 5 eq), gradually warmed to room temperature, stirred overnight to give a thick reaction mixture, slowly poured into ice water, ethyl acetate (150 mL × 3), combined organic layer, brine The mixture was washed with anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product, which was purified by column chromatography (PE/EA = 1/1) to give product (4-methylsulfonylbenzyl alcohol) 9.2 g. 81.9%.
1H NMR(500MHz，DMSO-d6):δ(ppm)7.888-7.905(2H，d)，7.543-7.560(2H，d)，4.806(2H，s)，3.035(3H，s)。 1 H NMR (500 MHz, DMSO-d 6 ): δ (ppm) 7.88-7.905 (2H, d), 7.453-7.560 (2H, d), 4.068 (2H, s), 3.035 (3H, s).
步骤2： 4-甲砜基苄氯的制备Step 2: Preparation of 4-methylsulfonylbenzyl chloride
对甲砜基苄醇(11.2g,60.0mmol,1eq)溶于干燥的70mL二氯甲烷中，降温至0℃，加入三乙胺(24.3g,240.0mmol,4eq),缓慢滴加氯化亚砜(14.3g,120.0mmol,2eq)，滴毕，逐渐 升温至回流，反应3h，停止加热冷却至室温，滴加50mL水淬灭反应，分层。有机层用无水硫酸钠干燥、减压浓缩除去有机溶剂得粗产物，经硅胶柱层析(PE/EA＝4/3)得产物(4-甲砜基苄氯)11.2g,产率91.3％。p-Methylsulfonylbenzyl alcohol (11.2 g, 60.0 mmol, 1 eq) was dissolved in dry 70 mL dichloromethane, cooled to 0 ° C, triethylamine (24.3 g, 240.0 mmol, 4 eq) Sulfone (14.3g, 120.0mmol, 2eq), dripped, gradually The temperature was raised to reflux, the reaction was carried out for 3 h, the heating was stopped and cooled to room temperature, and 50 mL of water was added dropwise to quench the reaction, and the layers were separated. The organic layer was dried over anhydrous sodium sulfate (MgSO4) (EtOAcjjjjjjj %.
步骤3：(1-(4-(甲磺酰基)苄基)-1H-咪唑-4，5-二甲酸二甲酯的制备Step 3: Preparation of (1-(4-(methylsulfonyl)benzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester
游离的1H-咪唑-4，5-二甲酸二甲酯(9.2g，50mmol，1eq)、对甲砜基苄氯(20.5g，100mmol，2eq)和碳酸钾(17.3g，125.0mmol，2.5eq)依次溶于50mL N,N-二甲基甲酰胺中，120℃搅拌反应20h，TLC监控反应至终点。反应液冷却至室温，加入150mL水，乙酸乙酯萃取(100mL×3)，合并乙酸乙酯层，依次水洗(100mL×3)和饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得产物为无色透明液体((1-(4-(甲磺酰基)苄基)-1H-咪唑-4，5-二甲酸二甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (9.2 g, 50 mmol, 1 eq), p-methylsulfonylbenzyl chloride (20.5 g, 100 mmol, 2 eq) and potassium carbonate (17.3 g, 125.0 mmol, 2.5 eq The solution was dissolved in 50 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 20 h, and the reaction was monitored by TLC to the end. The reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL×3), and the ethyl acetate layer was combined, washed with water (100 mL×3) and brine (100 mL×2), dried over anhydrous sodium sulfate. Concentrated under reduced pressure to remove the organic solvent to give the product as a colorless transparent liquid ((1-(4-(methylsulfonyl)benzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl)) .
步骤4：(1-(4-(甲磺酰基)苄基)-1H-咪唑-4，5-二酰胺的制备Step 4: Preparation of (1-(4-(methylsulfonyl)benzyl)-1H-imidazole-4,5-diamide
步骤3所得的产物(1-(4-(甲磺酰基)苄基)-1H-咪唑-4，5-二甲酸二甲酯溶于约50ml氨水中，45℃搅拌反应过夜，得白色混悬液，过滤，滤饼用水洗涤(10mL×3)，50℃减压干燥，得产物((1-(4-(甲磺酰基)苄基)-1H-咪唑-4，5-二酰胺)6.6g。最后两步总产率41％。The product obtained in the step 3 (1-(4-(methylsulfonyl)benzyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester is dissolved in about 50 ml of aqueous ammonia, and stirred at 45 ° C overnight to obtain a white suspension. The liquid was filtered, and the filter cake was washed with water (10 mL×3), and dried under reduced pressure at 50 ° C to give the product ((1-(4-(methylsulfonyl)benzyl)-1H-imidazole-4,5-diamide) 6.6 g. The total yield of the last two steps was 41%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.666(1H，s)，8.156(1H，s)，8.032(1H，s)，7.868-7.885(2H，d)，7.838(1H，s)，7.527(1H，s)，7.337-7.354(2H，d)，5.823(2H，s),3.178(3H，s)。 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 10.666 (1H, s), 8.156 (1H, s), 8.032 (1H, s), 7.868-7.885 (2H, d), 7.838 (1H, s), 7.527 (1H, s), 7.337-7.354 (2H, d), 5.823 (2H, s), 3.178 (3H, s).
实施例12 1-(4-(4-氯苯氧基)苯基)-1H-咪唑-4，5-二酰胺(15167)的制备Example 12 Preparation of 1-(4-(4-chlorophenoxy)phenyl)-1H-imidazole-4,5-diamide (15167)
Figure PCTCN2016077918-appb-000015
Figure PCTCN2016077918-appb-000015
步骤1： 4-(4-氯苯氧基)苯甲醛的制备Step 1: Preparation of 4-(4-chlorophenoxy)benzaldehyde
对氟苯甲醛(18.6g，150mmol，1eq)与对氯苯酚(19.3g，150mmol，1eq)及碳酸钾(51.8g，375mmol，2.5eq)溶于100mL N,N-二甲基甲酰胺中，70℃搅拌反应5小时，TLC监控反应至终点，冷却至室温，加入200mL水稀释，乙酸乙酯萃取(200mL×3)，合并有机层、饱和食盐水洗涤、无水硫酸钠干燥、减压浓缩得浸膏状粗产物，硅胶柱层析分离(PE/EA＝4/1)得产物(4-(4-氯苯氧基)苯甲醛)，该产物直接进行下一步反应。p-Fluorobenzaldehyde (18.6 g, 150 mmol, 1 eq) was dissolved in 100 mL of N,N-dimethylformamide with p-chlorophenol (19.3 g, 150 mmol, 1 eq) and potassium carbonate (51.8 g, 375 mmol, 2.5 eq). The reaction was stirred at 70 ° C for 5 hours, and the reaction was cooled to EtOAc EtOAc (EtOAc) (EtOAc) The crude product was obtained as a crude product, which was purified by silica gel column chromatography (PE/EA=4/1) to give the product (4-(4-chlorophenoxy)benzaldehyde).
1H NMR(CDCl3，500MHz):δ(ppm)9.926(1H，s)，7.864-7.847(2H，d)，7.358-7.376(2H，d)，7.013-7.065(4H，m)。 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) 9.926 (1H, s), 7.864-7.847 (2H, d), 7.358-7.376 (2H, d), 7.013-7.065 (4H, m).
步骤2：(4-(4-氯苯氧基)苯基)甲醇的制备 Step 2: Preparation of (4-(4-chlorophenoxy)phenyl)methanol
4-(4-氯苯氧基)苯甲醛(11.6g，50mmol，1eq)溶于100mL四氢呋喃中，降温至0℃，分批加入氢化铝锂(11.6g，150mmol，3eq)，逐渐升温至室温，搅拌反应4h，TLC监控反应至终点，加入10H2O.H2SO4淬灭反应，再加入200mL乙酸乙酯搅拌约半小时，抽滤除去不溶物，滤液100mL水洗3遍、饱和食盐水洗涤、无水硫酸钠干燥、减压浓缩得产物((4-(4-氯苯氧基)苯基)甲醇)，直接进行下一步反应。4-(4-Chlorophenoxy)benzaldehyde (11.6 g, 50 mmol, 1 eq) was dissolved in 100 mL of tetrahydrofuran, cooled to 0 ° C, and lithium aluminum hydride (11.6 g, 150 mmol, 3 eq) was added portionwise and gradually warmed to room temperature The reaction was stirred for 4 h, and the reaction was quenched to the end by TLC. The reaction was quenched by the addition of 10H 2 OH 2 SO 4 , then ethyl acetate (200 mL) was added and stirred for about half an hour, and the insolubles were removed by suction filtration, and the filtrate was washed three times with water and washed with saturated brine. Drying over anhydrous sodium sulfate and concentrating under reduced pressure afforded ((4-(4-chlorophenoxy)phenyl)methanol).
步骤3： 1-氯-4-(4-(氯甲基)苯氧基)苯的制备Step 3: Preparation of 1-chloro-4-(4-(chloromethyl)phenoxy)benzene
(4-(4-氯苯氧基)苯基)甲醇(12g，51.3mmol，1eq)溶于100mL二氯甲烷中，降温至0℃，加入三乙胺(20.8g,205.2mmol,4eq)，然后缓慢滴加氯化亚砜(12.2g,102.6mmol,2eq)，滴毕升温至室温，搅拌反应3小时，TLC监控反应至终点，加50mL水淬灭反应，反应液分层，有机层用水洗涤(50mL×3)、饱和食盐水洗涤、无水硫酸钠干燥、减压浓缩得浸膏状产物(1-氯-4-(4-(氯甲基)苯氧基)苯)直接用于下一步反应。(4-(4-Chlorophenoxy)phenyl)methanol (12 g, 51.3 mmol, 1 eq) was dissolved in 100 mL dichloromethane, EtOAc (EtOAc) Then, thionyl chloride (12.2 g, 102.6 mmol, 2 eq) was slowly added dropwise, and the mixture was warmed to room temperature, stirred for 3 hours, and the reaction was monitored by TLC to the end. The reaction was quenched with 50 mL of water and the mixture was partitioned. Washing (50 mL × 3), washing with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to give a product (1-chloro-4-(4-(chloromethyl)phenoxy)benzene) The next step is to react.
步骤4： 1-(4-(4-氯苯氧基)苯基)-1H-咪唑-4，5-二甲酸甲酯的制备Step 4: Preparation of methyl 1-(4-(4-chlorophenoxy)phenyl)-1H-imidazole-4,5-dicarboxylate
游离的1H-咪唑-4，5-二甲酸二甲酯(9.2g，50mmol，1eq)、1-氯-4(4-(氯甲基)苯氧基)苯(20.5g，100mmol，2eq)和碳酸钾(17.3g，125.0mmol，2.5eq)依次溶于60mL N,N-二甲基甲酰胺中，120℃搅拌反应20h，TLC监控反应至终点。反应液冷却至室温，加入150mL水，乙酸乙酯萃取(100mL×3)，合并乙酸乙酯层，依次水洗(100mL×3)和饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，硅胶柱层析分离(PE/EA＝4/3)得产物(1-(4-(4-氯苯氧基)苯基)-1H-咪唑-4，5-二甲酸甲酯)，直接用于下一步反应。Free 1H-imidazole-4,5-dicarboxylic acid dimethyl ester (9.2 g, 50 mmol, 1 eq), 1-chloro-4(4-(chloromethyl)phenoxy)benzene (20.5 g, 100 mmol, 2 eq) Potassium carbonate (17.3 g, 125.0 mmol, 2.5 eq) was sequentially dissolved in 60 mL of N,N-dimethylformamide, and the reaction was stirred at 120 ° C for 20 h, and the reaction was monitored by TLC to the end. The reaction mixture was cooled to room temperature, and then added with 150 mL of water and ethyl acetate (100 mL×3), and the ethyl acetate layer was combined, washed with water (100 mL×3) and brine (100 mL×2), dried over anhydrous sodium sulfate. The product (1-(4-(4-chlorophenoxy)phenyl)-1H-imidazole-4,5-dicarboxylic acid methyl ester) was obtained by silica gel column chromatography (PE/EA=4/3). In the next step.
1H NMR(CDCl3，500MHz):δ(ppm)7.734(1H，s)，7.287-7.305(2H，d)，7.161-7.178(2H，d)，6.920-6.912(4H，m)，5.389(2H，s)，3.920(3H，s)，3.873(3H，s)。 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) 7.734 (1H, s), 7.287-7.305 (2H, d), 7.161-7.178 (2H, d), 6.920-6.912 (4H, m), 5.389 ( 2H, s), 3.920 (3H, s), 3.873 (3H, s).
步骤5： 1-(4-(4-氯苯氧基)苯基)-1H-咪唑-4，5-二酰胺的制备Step 5: Preparation of 1-(4-(4-chlorophenoxy)phenyl)-1H-imidazole-4,5-diamide
1-(4-(4-氯苯氧基)苯基)-1H-咪唑-4，5-二甲酸甲酯(10g，25mmol，1eq)加入到60ml氨水中，50℃搅拌10小时，有白色固体生成，得白色混悬液，过滤，滤饼用水洗涤(10mL×3)，50℃减压干燥，得产物(1-(4-(4-氯苯氧基)苯基)-1H-咪唑-4，5-二酰胺)11.8g。最后两步反应总产率是63.7％。Methyl 1-(4-(4-chlorophenoxy)phenyl)-1H-imidazole-4,5-dicarboxylate (10 g, 25 mmol, 1 eq) was added to 60 ml of aqueous ammonia and stirred at 50 ° C for 10 hours with white The solid was formed to give a white suspension, which was filtered, washed with water (10mL×3), and dried under reduced pressure at 50 ° C to give the product (1-(4-(4-chlorophenoxy)phenyl)-1H-imidazole -4,5-diamide) 11.8 g. The total yield of the last two steps was 63.7%.
1H NMR(500MHz，DMSO-d6):δ(ppm)10.663(1H，s)，8.130(1H，s)，8.026(1H，s)，7.834(1H，s)，7.831(1H，s)，7.400-7.589(2H，d)，7.229-7.252(2H，d)，6.982-7.016(4H，m)，5.707(2H，s)。 1 H NMR (500MHz, DMSO- d 6): δ (ppm) 10.663 (1H, s), 8.130 (1H, s), 8.026 (1H, s), 7.834 (1H, s), 7.831 (1H, s) , 7.400-7.589 (2H, d), 7.229-7.252 (2H, d), 6.982-7.016 (4H, m), 5.707 (2H, s).
实施例13 N-苄基咪酰胺衍生物对硫酸粘杆菌素敏感大肠杆菌抑制活性的协同增效作用。 Example 13 Synergistic effect of N-benzylmidamide derivatives on colistin-sensitive Escherichia coli inhibitory activity.
利用试管二倍稀释法测试硫酸粘杆菌素和N-苄基咪酰胺衍生物对硫酸粘杆菌素耐药大肠杆菌(对硫酸粘杆菌素耐药，MIC值大于4.0ppm)的体外最小抑制浓度(MIC)，同时测试在培养时培养液中分别含有100.0ppmN-苄基咪酰胺衍生物时硫酸粘杆菌素对相应菌株的体外最小抑制浓度(其是以N-苄基咪酰胺衍生物和硫酸粘杆菌素联用作为测试药物，测试时，N-苄基咪酰胺衍生物的浓度固定为100.0ppm，而硫酸粘杆菌素进行梯度稀释，其目的是测试在含有100.0ppmN-苄基咪酰胺衍生物时硫酸粘杆菌素对相应菌株的体外最小抑制浓度)。结果显示，所有试验菌株对硫酸粘杆菌素耐药而所有试验的N-苄基咪酰胺衍生物对耐药大肠杆菌无抑制活性(表1)；而在含有100.0ppmN-苄基咪酰胺衍生物的测试组硫酸粘杆菌素对相应试验菌株的最小抑制浓度均有不同程度的降低，降低50-100倍(表2)。The in vitro minimum inhibitory concentration of colistin sulfate and N-benzylimimidamide derivatives against colistin-resistant Escherichia coli (resistant to colistin sulfate, MIC value greater than 4.0 ppm) was tested by tube double dilution method ( MIC), while testing the minimum inhibitory concentration of colistin sulfate to the corresponding strain in the culture medium containing 100.0 ppm of N-benzylimicuramide derivative in culture (which is N-benzylmidamide derivative and sulfuric acid) The combination of bacillus and bacillus was used as a test drug. When tested, the concentration of the N-benzylmidamide derivative was fixed at 100.0 ppm, and the colistin sulfate was subjected to gradient dilution for the purpose of testing the derivative containing 100.0 ppm of N-benzylimidamide. The minimum inhibitory concentration of colistin sulfate to the corresponding strain in vitro). The results showed that all the test strains were resistant to colistin sulfate and all the tested N-benzylimimidamide derivatives had no inhibitory activity against resistant Escherichia coli (Table 1); while containing 100.0 ppm of N-benzylmidamide derivatives The test group of colistin sulfate had different degrees of reduction in the minimum inhibitory concentration of the corresponding test strains, which was reduced by 50-100 times (Table 2).
表1 不同N-苄基咪酰胺衍生物对硫酸粘杆菌素耐药大肠杆菌的体外最小抑制浓度(MIC，ppm)Table 1 In vitro minimum inhibitory concentration (MIC, ppm) of different N-benzylimimidamide derivatives against colistin sulfate-resistant Escherichia coli
  大肠杆菌2G-7Escherichia coli 2G-7 大肠杆菌5F-1Escherichia coli 5F-1 大肠杆菌4D-6E. coli 4D-6 大肠杆菌3H-4Escherichia coli 3H-4
硫酸粘杆菌素Colistin sulfate 6464 128128 256256 6464
1503115031 >800>800 >800>800 >800>800 >800>800
1503715037 >800>800 >800>800 >800>800 >800>800
1504115041 >800>800 >800>800 >800>800 >800>800
1504215042 >800>800 >800>800 >800>800 >800>800
1504515045 >800>800 >800>800 >800>800 >800>800
1504615046 >800>800 >800>800 >800>800 >800>800
1505115051 >800>800 >800>800 >800>800 >800>800
1505315053 >800>800 >800>800 >800>800 >800>800
1505415054 >800>800 >800>800 >800>800 >800>800
1505615056 >800>800 >800>800 >800>800 >800>800
1516715167 >800>800 >800>800 >800>800 >800>800
表2 N-苄基咪酰胺衍生物存在下硫酸粘杆菌素对硫酸粘杆菌素耐药大肠杆菌的体外最小抑制浓度(MIC，ppm)Table 2 In vitro minimum inhibitory concentration (MIC, ppm) of colistin sulfate resistant to colistin sulfate-resistant Escherichia coli in the presence of N-benzylmidamide derivative
Figure PCTCN2016077918-appb-000016
Figure PCTCN2016077918-appb-000016
Figure PCTCN2016077918-appb-000017
Figure PCTCN2016077918-appb-000017
实施例14 N-苄基咪酰胺衍生物对硫酸粘杆菌素敏感大肠杆菌抑制活性的协同增效作用。Example 14 Synergistic effect of N-benzylmidamide derivatives on colistin-sensitive Escherichia coli inhibitory activity.
利用试管二倍稀释法测试硫酸粘杆菌素和N-苄基咪酰胺衍生物对大肠杆菌(对硫酸粘杆菌素敏感，MIC值小于4.0ppm)的体外最小抑制浓度(MIC)，同时测试在培养中分别含有100.0ppmN-苄基咪酰胺衍生物时硫酸粘杆菌素对相应菌株的体外最小抑制浓度(其是以N-苄基咪酰胺衍生物和硫酸粘杆菌素联用作为测试药物，测试时，N-苄基咪酰胺衍生物的浓度固定为100.0ppm，而硫酸粘杆菌素进行梯度稀释，其目的是测试在含有100.0ppmN-苄基咪酰胺衍生物时硫酸粘杆菌素对相应菌株的体外最小抑制浓度)。结果如表3所示，从表3可以看出，所有试验菌株对硫酸粘杆菌素敏感而所有试验用N-苄基咪酰胺衍生物对试验菌株无抑制活性。而含有100.0ppmN-苄基咪酰胺衍生物的测试组硫酸粘杆菌素对相应试验菌株的最小抑制浓度有不同程度的降低，约2-4倍(表4)。The in vitro minimum inhibitory concentration (MIC) of colistin sulfate and N-benzylmidamide derivatives against Escherichia coli (sensitive to colistin sulfate, MIC value less than 4.0 ppm) was tested by tube double dilution method, and tested in culture. The minimum inhibitory concentration of colistin sulfate to the corresponding strain in the case of containing 100.0 ppm of N-benzylimimidamide derivative respectively (the combination of N-benzylmidamide derivative and colistin sulfate as test drug) The concentration of the N-benzylmidamide derivative was fixed at 100.0 ppm, and the colistin sulfate was subjected to a gradient dilution for the purpose of testing the coagulizin sulfate for the corresponding strain in the case of containing 100.0 ppm of the N-benzylimimidamide derivative. Minimum inhibitory concentration). The results are shown in Table 3. As can be seen from Table 3, all the test strains were sensitive to colistin sulfate and all the test N-benzylmidamide derivatives had no inhibitory activity against the test strain. The test group containing 100.0 ppm of N-benzylimimidamide derivative showed a minimum reduction in the minimum inhibitory concentration of the corresponding test strain, about 2-4 times (Table 4).
表3 N-苄基咪酰胺衍生物对硫酸粘杆菌素敏感大肠杆菌的体外最小抑制浓度(MIC，ppm)Table 3 In vitro minimum inhibitory concentration (MIC, ppm) of N-benzylmidamide derivatives against colistin sulfate-sensitive Escherichia coli
  大肠杆菌3Y-9E. coli 3Y-9 大肠杆菌2S-19E. coli 2S-19 大肠杆菌5W-7E. coli 5W-7 大肠杆菌3B-24E. coli 3B-24
硫酸粘杆菌素Colistin sulfate 2.02.0 2.02.0 1.01.0 2.02.0
1503115031 >800>800 >800>800 >800>800 >800>800
1503715037 >800>800 >800>800 >800>800 >800>800
1504115041 >800>800 >800>800 >800>800 >800>800
1504215042 >800>800 >800>800 >800>800 >800>800
1504515045 >800>800 >800>800 >800>800 >800>800
1504615046 >800>800 >800>800 >800>800 >800>800
1505115051 >800>800 >800>800 >800>800 >800>800
1505315053 >800>800 >800>800 >800>800 >800>800
1505415054 >800>800 >800>800 >800>800 >800>800
1505615056 >800>800 >800>800 >800>800 >800>800
1516715167 >800>800 >800>800 >800>800 >800>800
表4 N-苄基咪酰胺衍生物存在下硫酸粘杆菌素对硫酸粘杆菌素敏感大肠杆菌的体外最小抑制浓度(MIC，ppm)Table 4 In vitro minimum inhibitory concentration (MIC, ppm) of colistin sulfate-sensitive Escherichia coli in the presence of N-benzylmidamide derivatives
Figure PCTCN2016077918-appb-000018
Figure PCTCN2016077918-appb-000018
实施例15 不同浓度N-苄基咪酰胺衍生物(15037)对不同种类的硫酸粘杆菌素耐药菌株的耐药性逆转作用。Example 15 Reversal of drug resistance of different types of colistin sulfate resistant strains with different concentrations of N-benzylmidamide derivatives (15037).
利用试管二倍稀释法测试硫酸粘杆菌素和N-苄基咪酰胺衍生物(15037)对不同种类硫酸 粘杆菌素耐药的革兰氏阴性细菌(对硫酸粘杆菌素耐药，MIC值大于4.0ppm)的体外最小抑制浓度，同时测试在培养时培养液中分别含有不同浓度N-苄基咪酰胺衍生物(15037)时硫酸粘杆菌素对相应菌株的体外最小抑制浓度。结果显示，所有试验菌株对硫酸粘杆菌素均耐药；N-苄基咪酰胺衍生物(15037)对所有测试菌株无抑制活性；而含有N-苄基咪酰胺衍生物(15037)的测试组硫酸粘杆菌素对相应菌株的最小抑制浓度均有不同程度的降低，呈明显的剂量效应(表5)。Tests for different types of sulfuric acid by using the test tube double dilution method for colistin sulfate and N-benzylmidamide derivatives (15037) The minimum inhibitory concentration of colistin-resistant Gram-negative bacteria (resistant to colistin sulfate, MIC value greater than 4.0 ppm), and the test also contained different concentrations of N-benzylmidamide in the culture medium. The minimum inhibitory concentration of colistin sulfate to the corresponding strain in the derivative (15037). The results showed that all the test strains were resistant to colistin sulfate; the N-benzylmidamide derivative (15037) had no inhibitory activity against all test strains; and the test group containing the N-benzylmidamide derivative (15037) The minimum inhibitory concentration of colistin sulfate on the corresponding strains was reduced to varying degrees, showing a significant dose effect (Table 5).
表5 不同浓度的15037存在下硫酸粘杆菌素对不同种类细菌的体外最小抑制浓度(MIC，ppm)Table 5 In vitro minimum inhibitory concentration (MIC, ppm) of colistin sulfate for different types of bacteria in the presence of different concentrations of 15037
Figure PCTCN2016077918-appb-000019
Figure PCTCN2016077918-appb-000019
实施例16 N-苄基咪酰胺衍生物与硫酸粘杆菌素在断奶仔猪料中的协同应用效果。Example 16 Synergistic application effect of N-benzylmidamide derivative and colistin sulfate in weaned piglets.
130头28日龄体重相近的杜长大三元杂瘦肉型断奶仔猪分成13组，每组10头。各组在不含抗生素的教槽料中添加硫酸粘杆菌素和/或不同种类的N-苄基咪酰胺衍生物，试验期间自由采食和饮水，统计10天内各试验组试验猪的增重、饲料报酬和腹泻率。结果显示(表6)，在断奶仔猪料中仅添加硫酸粘杆菌素不能有效降低试验猪的腹泻率，对增重和饲料报酬未见明显改善；而同时添加N-苄基咪酰胺衍生物和硫酸粘杆菌素的测试组可不同程度降低试验组的腹泻率并对生产性能有不同程度改善。130 pairs of 28-day-old Du Changda ternary miscellaneous meat-type weaned piglets were divided into 13 groups of 10 heads each. Each group added colistin sulfate and/or different kinds of N-benzylmidamide derivatives to the non-antibiotic-containing feedstock. During the test period, they were fed ad libitum and water, and the weight gain of the test pigs in each test group was counted within 10 days. , feed compensation and diarrhea rate. The results showed (Table 6) that the addition of colistin sulfate to the weaned piglets did not effectively reduce the diarrhea rate of the test pigs, and did not significantly improve the weight gain and feed compensation; while adding N-benzylmidamide derivatives and The test group of colistin sulfate reduced the diarrhea rate of the test group to varying degrees and improved the production performance to varying degrees.
表6 N-苄基咪酰胺衍生物和硫酸粘杆菌素在断奶仔猪料中的协同应用效果Table 6 Synergistic effect of N-benzylmidamide derivatives and colistin sulfate in weaned piglets
Figure PCTCN2016077918-appb-000020
Figure PCTCN2016077918-appb-000020
Figure PCTCN2016077918-appb-000021
Figure PCTCN2016077918-appb-000021
实施例17 N-苄基咪酰胺衍生物(15037)和硫酸粘杆菌素在断奶仔猪料中协同应用的效果。Example 17 Effect of synergistic application of N-benzylmidamide derivative (15037) and colistin sulfate in weaned piglets.
120头28日龄体重相近的杜长大三元杂瘦肉型断奶仔猪分12组，每组10头。各组在不含抗生素的教槽料中添加硫酸粘杆菌素和/或N-苄基咪酰胺衍生物(15037)。试验期间自由采食和饮水，统计10天内各试验组试验猪的增重、饲料报酬和腹泻率。结果显示在断奶仔猪料中仅添加硫酸粘杆菌素或N-苄基咪酰胺衍生物(15037)均不能有效降低试验猪的腹泻率，对增重和饲料报酬未见明显改善；而添加硫酸粘杆菌素和N-苄基咪酰胺衍生物(15037)试验组(组10、11和12)可降低腹泻率，平均日增重和饲料报酬均显著改善(表7)。120 pairs of 28-day-old Du Changda ternary miscellaneous meat-type weaned piglets were divided into 12 groups of 10 heads each. Each group added colistin sulfate and/or N-benzylmidamide derivative (15037) to the antibiotic-free feedstock. During the test period, children were fed ad libitum and water, and the weight gain, feed remuneration and diarrhea rate of the test pigs in each test group were counted within 10 days. The results showed that the addition of colistin sulfate or N-benzylmidamide derivative (15037) in the weaned piglets could not effectively reduce the diarrhea rate of the test pigs, and did not significantly improve the weight gain and feed compensation; The bacteriocin and N-benzylmidamide derivatives (15037) test groups (groups 10, 11 and 12) reduced diarrhea rates, with a significant improvement in average daily gain and feed remuneration (Table 7).
表7 N-苄基咪酰胺衍生物(15037)和硫酸粘杆菌素在断奶仔猪料中的应用效果Table 7 Application effect of N-benzylmidamide derivative (15037) and colistin sulfate in weaned piglets
Figure PCTCN2016077918-appb-000022
Figure PCTCN2016077918-appb-000022
Figure PCTCN2016077918-appb-000023
Figure PCTCN2016077918-appb-000023
实施例18 不同剂量N-苄基咪酰胺衍生物(15037)和硫酸粘杆菌素在断奶仔猪料中协同应用的效果。Example 18 Effect of synergistic application of different doses of N-benzylmidamide derivative (15037) and colistin sulfate in weaned piglets.
90头28日龄体重相近的杜长大三元杂瘦肉型断奶仔猪分9组，每组10头。各组在不含抗生素的教槽料中添加硫酸粘杆菌素和/或不同剂量的N-苄基咪酰胺衍生物(15037)。试验期间自由采食和饮水，统计10天内各试验组试验猪的增重、饲料报酬和腹泻率。结果显示在断奶仔猪料中仅添加硫酸粘杆菌素或N-苄基咪酰胺衍生物(15037)均对试验猪的腹泻率，增重和饲料报酬改善有限。而同时添加硫酸粘杆菌素和N-苄基咪酰胺衍生物(15037)协同应用时，对腹泻率和生产性能均有明显改改善，且呈明显的剂量效应，随着N-苄基咪酰胺衍生物(15037)剂量的增加，效果越明显(表8)。90 pairs of 28-day-old Du Changda ternary heterozygous weaned piglets were divided into 9 groups, 10 in each group. Each group added colistin sulfate and/or different doses of N-benzylmidamide derivative (15037) to the antibiotic-free channel. During the test period, children were fed ad libitum and water, and the weight gain, feed remuneration and diarrhea rate of the test pigs in each test group were counted within 10 days. The results showed that the addition of colistin sulfate or N-benzylmidamide derivative (15037) to the weaned piglets had limited improvement in diarrhea rate, weight gain and feed compensation. At the same time, the addition of colistin sulfate and N-benzylmidamide derivative (15037) showed significant improvement in diarrhea rate and production performance, and showed obvious dose effect, along with N-benzylmidamide. The increase in the dose of the derivative (15037) was more pronounced (Table 8).
表8 不同剂量的15037和硫酸粘杆菌素在断奶仔猪料中的协同应用效果Table 8 Synergistic effects of different doses of 15037 and colistin sulfate in weaned piglets
Figure PCTCN2016077918-appb-000024
Figure PCTCN2016077918-appb-000024
Figure PCTCN2016077918-appb-000025
Figure PCTCN2016077918-appb-000025
实施例19 N-苄基咪酰胺衍生物(15037)与硫酸粘杆菌素在鸡料中的联合应用效果。Example 19 The combined use of N-benzylmidamide derivative (15037) and colistin sulfate in chicken feed.
800羽1日龄快大黄羽肉鸡随机分成4个试验组，每个试验组包含4个平行试验组，每个平行试验组为50羽，并分别在每组饲料中添加硫酸粘杆菌素或/和N-苄基咪酰胺衍生物(15037)。试验期笼养，自由采食和自由饮水，试验期共28天。试验结果显示，N-苄基咪酰胺衍生物(15037)与硫酸粘杆菌素对试验鸡的增重及饲料报酬表现出协同增效的效应(表9)。800 feather 1 day old fast yellow broilers were randomly divided into 4 test groups, each test group consisted of 4 parallel test groups, 50 in each parallel test group, and colistin sulfate was added to each group of feeds. And N-benzylmidamide derivatives (15037). During the test period, they were caged, free to eat and free to drink. The test period was 28 days. The test results showed that the N-benzylmidamide derivative (15037) and colistin sulfate showed synergistic effects on the weight gain and feed remuneration of the test chickens (Table 9).
表9 N-苄基咪酰胺衍生物(15037)与硫酸粘杆菌素在肉鸡料中的联合应用效果Table 9 Effect of combined application of N-benzylmidamide derivative (15037) and colistin sulfate in broiler feed
Figure PCTCN2016077918-appb-000026
Figure PCTCN2016077918-appb-000026
实施例20 N-苄基咪酰胺衍生物(15037)与硫酸粘杆菌素在肉鸭料中的联合应用效果。Example 20 The combined use effect of N-benzylmidamide derivative (15037) and colistin sulfate in meat duck feed.
800羽1日龄樱桃谷肉鸭按表10随机分成4个试验组，每个试验组包含4个平行试验组，每个平行试验组为50羽，并分别在每组饲料中添加硫酸粘杆菌素或/和N-苄基咪酰胺衍生物(15037)。试验期笼养，自由采食和自由饮水。试验期共21天。试验结果显示，N-苄基咪酰胺衍生物(15037)与硫酸粘杆菌素对试验鸭的增重及饲料报酬表现出协同增效的效应(表10)。 800 feathers of 1 day old Cherry Valley meat ducks were randomly divided into 4 test groups according to Table 10. Each test group contained 4 parallel test groups, 50 in each parallel test group, and added Lactobacillus sulfate in each group of feeds. Or / and N-benzylmidamide derivatives (15037). Cage in the trial period, free access to food and free drinking water. The trial period was 21 days. The test results showed that the N-benzylmidamide derivative (15037) and colistin sulfate showed synergistic effects on the weight gain and feed remuneration of the test ducks (Table 10).
表10 N-苄基咪酰胺衍生物(15037)与硫酸粘杆菌素在肉鸭料中的联合应用效果Table 10 Effect of combined application of N-benzylmidamide derivative (15037) and colistin sulfate in meat duck feed
Figure PCTCN2016077918-appb-000027
Figure PCTCN2016077918-appb-000027

Claims (12)

  1. N-苄基咪酰胺衍生物用作多粘菌素类抗生素协同增效剂的应用，其特征在于，所述的N-苄基咪酰胺衍生物的结构如式(Ⅰ)所示：The use of an N-benzylmidamide derivative as a polymyxin antibiotic synergist, characterized in that the structure of the N-benzylmidamide derivative is as shown in the formula (I):
    Figure PCTCN2016077918-appb-100001
    Figure PCTCN2016077918-appb-100001
    其中，R1和R2分别为H、卤素、NO2、S(＝O)2CH3、O-Ph、O-C1-8直链或支链烷基、C1-8直链或支链烷基、或、C1-8直链或支链的卤代烷基；Wherein, R 1 and R 2 are independently H, halo, NO 2, S (= O ) 2 CH 3, O-Ph, OC 1-8 straight-chain or branched-chain alkyl group, C 1-8 straight-chain or branched-chain An alkyl group, or a C 1-8 linear or branched haloalkyl group;
    所述的卤素为F、Cl、Br或I；The halogen is F, Cl, Br or I;
    所述的O-Ph中的苯基是指未被取代或被1，2，3，4个取代基取代的苯基，所述的取代基选自F、Cl、Br、I、C1-8直链或支链烷基。The phenyl group in the O-Ph means a phenyl group which is unsubstituted or substituted with 1, 2, 3, 4 substituents selected from the group consisting of F, Cl, Br, I, C 1- 8 linear or branched alkyl groups.
  2. 根据权利要求1所述的应用，其特征在于，所述的N-苄基咪酰胺衍生物，其R1为H，R2为卤素。The use according to Claim 1, characterized in that the N-benzylmidamide derivative has R 1 as H and R 2 as a halogen.
  3. 根据权利要求2所述的应用，其特征在于，所述的N-苄基咪酰胺衍生物，其具有式(Ⅱ)所示的结构：The use according to Claim 2, characterized in that the N-benzylmidamide derivative has the structure represented by the formula (II):
    Figure PCTCN2016077918-appb-100002
    Figure PCTCN2016077918-appb-100002
  4. 根据权利要求1所述的应用，其特征在于，所述的多粘菌素类抗生素为多粘菌素A、多粘菌素B、多粘菌素C、多粘菌素D或多粘菌素E。The use according to claim 1, wherein the polymyxin antibiotic is polymyxin A, polymyxin B, polymyxin C, polymyxin D or polymyxobacteria. Prime E.
  5. 根据权利要求1-4任一项所述的应用，其特征在于，所述的N-苄基咪酰胺衍生物作为多粘菌素类抗生素协同增效剂的应用是用于制备抗多粘菌素类抗生素敏感菌或耐药菌的药物。 The use according to any one of claims 1 to 4, characterized in that the N-benzylmidamide derivative is used as a synergistic synergist for polymyxins for the preparation of anti-polymyxobacteria A drug that is a sensitive antibiotic or a drug resistant bacteria.
  6. 根据权利要求5所述的应用，其特征在于，所述N-苄基咪酰胺衍生物使用剂量为10-200ppm。The use according to Claim 5, characterized in that the N-benzylmidamide derivative is used in a dose of from 10 to 200 ppm.
  7. 根据权利要求1-4任一项所述的应用，其特征在于，所述的N-苄基咪酰胺衍生物作为硫酸粘杆菌素协同增效剂的应用是用于制备动物生长促进剂。The use according to any one of claims 1 to 4, characterized in that the use of the N-benzylmidamide derivative as a synergist for colistin sulfate is for the preparation of an animal growth promoter.
  8. 根据权利要求7所述的应用，其特征在于，所述的N-苄基咪酰胺衍生物在饲料中的添加剂量是10-500ppm。The use according to claim 7, characterized in that the amount of the N-benzylmidamide derivative in the feed is from 10 to 500 ppm.
  9. 根据权利要求7所述的应用，其特征在于，所述的动物是畜禽。The use according to claim 7, wherein the animal is a livestock.
  10. 根据权利要求9所述的应用，其特征在于，所述的畜禽是各个生长阶段的鸡、鸭、鹅、鸽、鹌鹑、猪、牛、羊、马、兔、驴、鹿、狗、猫、狐、貂或貉。The use according to claim 9, wherein the livestock is chicken, duck, goose, pigeon, donkey, pig, cow, sheep, horse, rabbit, donkey, deer, dog, cat at various stages of growth. , fox, sly or sly.
  11. 根据权利要求1-4任一项所述的应用，其特征在于，所述的N-苄基咪酰胺衍生物作为多粘菌素类抗生素协同增效剂的应用是用于制备人用抗革兰氏阴性菌感染的药物。The use according to any one of claims 1 to 4, characterized in that the use of the N-benzylmidamide derivative as a synergistic synergist for polymyxins is for the preparation of artificial leather for human use. A drug that is infected with a gram-negative bacteria.
  12. 根据权利要求11所述的应用，其特征在于，所述的抗革兰氏阴性菌感染的药物是通过口服方式、注射方式或外用方式给药的抗革兰氏阴性菌感染的药物。 The use according to claim 11, wherein the drug against Gram-negative bacteria infection is a drug against Gram-negative infection which is administered orally, by injection or externally.
PCT/CN2016/077918 2015-12-29 2016-03-30 Application of n-benzylimidazolecarboxamide derivatives as synergist for polymyxin antibiotics WO2017113525A1 (en)

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CN115779068A (en) * 2022-12-29 2023-03-14 青岛农业大学 Synergistic agent for polymyxin antibiotics and application thereof

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