WO2023201712A1 - Clofoctol derivative, antibacterial medicament, preparation method therefor, and use thereof - Google Patents

Clofoctol derivative, antibacterial medicament, preparation method therefor, and use thereof Download PDF

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WO2023201712A1
WO2023201712A1 PCT/CN2022/088513 CN2022088513W WO2023201712A1 WO 2023201712 A1 WO2023201712 A1 WO 2023201712A1 CN 2022088513 W CN2022088513 W CN 2022088513W WO 2023201712 A1 WO2023201712 A1 WO 2023201712A1
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chlorphenol
derivative
group
atoms
antibacterial
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PCT/CN2022/088513
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French (fr)
Chinese (zh)
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林水木
刘寿平
刘家勇
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广州医科大学
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    • 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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C277/00—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C277/08—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups of substituted guanidines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/12—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
    • 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

Definitions

  • the present invention relates to the field of antibacterial, in particular to a chlorphenol derivative, an antibacterial drug, a preparation method and application.
  • Gram-negative bacteria have a special outer membrane composed of a lipopolysaccharide layer and membrane phospholipids, making it difficult for most antibacterial drugs to penetrate the bacterial cell outer membrane to exert effective antibacterial activity. Therefore, it is extremely important to develop new antibacterial drugs that can overcome or slow down the development of drug resistance, especially drugs against Gram-negative bacteria.
  • the present invention provides a chlorphenol derivative, which is highly efficient and low-toxic, has a broad antibacterial spectrum, fast sterilization speed, excellent antibacterial activity in vivo, good medicinal properties, and can effectively avoid the development of bacterial resistance.
  • the present invention is realized through the following technical solutions.
  • L 1 is selected from a linear alkyl group having 1 to 10 C atoms or a branched or cyclic alkyl group having 3 to 10 C atoms;
  • R1 is selected from a guanidine group, an amino group, an arginine group, a histidine group, a lysine group or a combination of these groups.
  • L 1 is selected from linear alkyl groups having 1 to 6 C atoms or branched or cyclic alkyl groups having 3 to 6 C atoms.
  • L 1 is selected from linear alkyl groups having 1 to 4 C atoms.
  • R 1 is selected from any one of the following groups:
  • the chlorphenol derivative is selected from the following structures:
  • the present invention also provides a method for preparing the chlorphenol derivative as described above, which includes the following steps:
  • the intermediate B is subjected to a dehydration condensation reaction to prepare the chlorphenol derivative.
  • the temperature of the Williamson synthesis reaction is 60°C to 70°C, and the time is 4h to 5h.
  • the present invention also provides the use of the above-mentioned chlorphenol derivatives in the preparation of antibacterial drugs.
  • the present invention also provides an antibacterial drug, the components of which include the above-mentioned chlorphenol derivatives, salts and pharmaceutically acceptable excipients.
  • the chlorphenol derivative of the present invention has the following beneficial effects:
  • the clofol derivative of the present invention uses clofol as a molecular skeleton, is an amphipathic cationic compound, has a novel membrane-targeted antibacterial mechanism, and broadens the antibacterial spectrum of clofol.
  • the chlorphenol derivatives of the present invention are effective against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, and Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii.
  • Klebsiella pneumoniae exhibit excellent in vitro and in vivo antibacterial activity, have good water solubility and excellent in vivo pharmacokinetic properties, and exhibit low cytotoxicity and hemolytic activity against mammalian cells.
  • Membrane selection It has high potency and good medicinal properties. This type of antibacterial drugs not only have strong broad-spectrum antibacterial activity, but can also overcome the development of bacterial resistance in laboratory simulated drug resistance studies.
  • Figure 1 shows the drug resistance research provided by the present invention; wherein, A represents the research on the resistance of FT09 and norfloxacin to Staphylococcus aureus ATCC29213, and B represents the research on the resistance of FT09 and amoxicillin to Escherichia coli ATCC25922. ;
  • Figure 2 is a cytotoxicity study provided by the present invention. where A represents the cytotoxicity of chlorphenol on mouse fibroblasts, and B represents the cytotoxicity of FT09 on mouse fibroblasts;
  • Figure 3 shows the effect of compound FT09 provided by the present invention on the integrity of the cell membrane of Staphylococcus aureus ATCC29213;
  • Figure 4 is an in vivo antibacterial activity study provided by the present invention; wherein, A represents the in vivo antibacterial activity study of FT09 and vancomycin against Staphylococcus aureus ATCC29213, and B represents the in vivo antibacterial activity study of FT09 and gatifloxacin against Pseudomonas aeruginosa ATCC9027. Antibacterial activity studies.
  • the elements carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds described in the present invention all include their isotope conditions, and the elements carbon, hydrogen, oxygen involved in the groups and compounds described in the present invention , sulfur or nitrogen are optionally further replaced by one or more of their corresponding isotopes.
  • alkyl refers to a saturated hydrocarbon containing primary (normal) carbon atoms, or secondary carbon atoms, or tertiary carbon atoms, or quaternary carbon atoms, or combinations thereof. Phrases containing this term, for example, "C 1 to C 9 alkyl” refer to alkyl groups containing 1 to 9 carbon atoms, and each occurrence may be independently C 1 alkyl, C 2 alkyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, C 6 alkyl, C 7 alkyl, C 8 alkyl, C 9 alkyl.
  • Suitable examples include, but are not limited to: methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ) , 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2 CH (CH 3 ) 2 ), 2-butyl (s-Bu, s-butyl, -CH (CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3
  • Ammonia refers to a derivative of ammonia having the structural characteristics of the formula -N(X) 2 , where each "X” is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted Cycloalkyl, substituted or unsubstituted heterocyclyl, etc.
  • Non-limiting types of amino groups include -NH 2 , -N(alkyl) 2 , -NH(alkyl), -N(cycloalkyl) 2 , -NH(cycloalkyl), -N(heterocyclyl) 2 , -NH (heterocyclyl), -N (aryl) 2 , -NH (aryl), -N (alkyl) (aryl), -N (alkyl) (heterocyclyl), -N (Cycloalkyl)(heterocyclyl), -N(aryl)(heteroaryl), -N(alkyl)(heteroaryl), etc.
  • the invention provides a chlorphenol derivative, the structure of which is shown in the general formula (1):
  • L 1 is selected from a linear alkyl group having 1 to 10 C atoms or a branched or cyclic alkyl group having 3 to 10 C atoms;
  • R1 is selected from a guanidine group, an amino group, an arginine group, a histidine group, a lysine group or a combination of these groups.
  • L 1 is selected from linear alkyl groups having 1 to 6 C atoms or branched or cyclic alkyl groups having 3 to 6 C atoms. More specifically, L 1 is selected from linear alkyl groups having 1 to 4 C atoms.
  • R 1 is selected from any one of the following groups:
  • chlorphenol derivative is selected from the following structures:
  • chlorphenol derivative is selected from the following structures:
  • connection site between amino acids is the amide bond by default.
  • the present invention also provides a preparation method of the above-mentioned chlorphenol derivative, which includes the following steps:
  • the intermediate B is subjected to a dehydration condensation reaction to prepare a chlorphenol derivative.
  • the temperature of the Williamson synthesis reaction is 60°C to 70°C, and the time is 4h to 5h.
  • the present invention also provides the use of the above-mentioned chlorphenol derivatives in the preparation of antibacterial drugs.
  • the present invention also provides an antibacterial drug, the components of which include the above-mentioned chlorphenol derivatives, salts and pharmaceutically acceptable excipients.
  • This embodiment provides a chlorphenol derivative FT07 and its preparation method.
  • the synthesis route is as follows:
  • HATU '-Tetramethylurea hexafluorophosphate
  • DIPEA N,N-diisopropylethylamine
  • L-arginine methyl ester dihydrochloride 157.3mg, 0.60mmol
  • This embodiment provides a chlorphenol derivative FT09 and its preparation method.
  • the synthesis route is as follows:
  • the reactants FT05 (80.0 mg, 0.18 mmol), HATU (168.9 mg, 0.44 mmol), DIPEA (154 ⁇ L, 0.88 mmol) and H-Arg-Arg-Arg-OMe ⁇ 4HCl (129.0 mg, 0.20 mmol) were synthesized according to the method of synthesizing FT07 Using this method, a white solid product FT09 (138.2 mg, 88%) was prepared.
  • This embodiment provides a chlorphenol derivative FT12 and its preparation method.
  • the synthesis route is as follows:
  • the reactants FT07 (64.4 mg, 0.11 mmol), HATU (120.9 mg, 0.33 mmol), DIPEA (111 ⁇ L, 0.65 mmol) and H-Arg-Arg-NH 2 ⁇ 3HCl (89.4 mg, 0.20 mmol) were synthesized according to the method for synthesizing FT07 Method, the yellow oily product FT12 (49.7 mg, 51%) was prepared.
  • This embodiment provides a chlorphenol derivative FT37 and its preparation method.
  • the synthesis route is as follows:
  • the reactants FT05 (75mg, 0.18mmol), HATU (202.1mg, 0.53mmol), DIPEA (185 ⁇ L, 1.06mmol) and H-Arg-Arg-OMe ⁇ 3HCl (116.4mg, 0.27mmol) were synthesized according to the method of synthesizing FT07,
  • the yellow-white solid product FT37 (103.6 mg, 80%) was prepared.
  • This embodiment provides a chlorphenol derivative FT40 and its preparation method.
  • the specific preparation steps are as follows:
  • This embodiment provides a chlorphenol derivative FT41 and its preparation method.
  • the synthesis route is as follows:
  • the reactants FT07 (50.3mg, 0.09mmol), HATU (99.0mg, 0.26mmol), DIPEA (91 ⁇ L, 0.52mmol) and H-Arg-Arg-Arg-OMe ⁇ 4HCl (86.9mg, 0.14mmol) were synthesized according to the method of synthesizing FT07 Using this method, the yellow oily product FT41 (38.6 mg, 42%) was prepared.
  • This embodiment provides a chlorphenol derivative FT42 and its preparation method.
  • the synthesis route is as follows:
  • the reactants FT37 (94.6mg, 0.13mmol), HATU (144.1mg, 0.38mmol), DIPEA (132 ⁇ L, 0.76mmol) and H-Arg-Arg-Arg-OMe ⁇ 4HCl (126.5mg, 0.20mmol) were synthesized according to the method of synthesizing FT07 Using this method, the yellow oily product FT42 (40.7 mg, 26%) was prepared.
  • This embodiment provides a chlorphenol derivative FT46 and its preparation method.
  • the preparation process is as follows:
  • reaction solution was stirred and reacted at room temperature for 24 hours. After the reaction is complete, the reaction solution is extracted twice with n-butanol, the organic phase is concentrated, dried, dissolved in dichloromethane (15 mL), and trifluoroacetic acid (3 mL) is added, mixed and stirred for 1 hour, and extracted twice with n-butanol. After several times, the organic phase was concentrated and purified using semi-preparative high-performance liquid phase separation. The product FT46 was prepared as a yellow oil (27.3 mg, 34%).
  • This embodiment provides a chlorphenol derivative FT49 and its preparation method.
  • the synthesis route is as follows:
  • the reactants FT05 (71.8 mg, 0.16 mmol), HATU (181.5 mg, 0.48 mmol), DIPEA (166 ⁇ L, 0.95 mmol) and H-Arg-Arg-NH 2 ⁇ 3HCl (104.6 mg, 0.24 mmol) were synthesized according to the method for synthesizing FT07 Method, a white solid product FT49 (86.3 mg, 74%) was prepared.
  • This embodiment provides a chlorphenol derivative FT60 and its preparation method.
  • the synthesis route is as follows:
  • the reactants FT59 (56.3 mg, 0.12 mmol), HATU (115.3 mg, 0.30 mmol), DIPEA (105 ⁇ L, 0.60 mmol) and H-Arg-Arg-Arg-OMe ⁇ 4HCl (90.8 mg, 0.14 mmol) were synthesized according to the method of synthesizing FT07 Using this method, the yellow oily product FT60 (96.4 mg, 76%) was prepared.
  • the antibacterial activity of the synthesized compounds was tested according to the broth dilution method specified by Clinical and Laboratory Standards Institute (CLSI) guidelines. Bacterial cells were inoculated onto Mueller-Hinton agar (MHA) plates and cultured overnight, and the bacterial cell concentration was adjusted to approximately 1 ⁇ 10 6 CFU/mL with PBS for preparation of bacterial suspension. The sample was first dissolved in DMSO/H 2 O to prepare a sample stock solution with a concentration of 1000 ⁇ g/mL (final concentration of DMSO ⁇ 2%), and then the stock solution was diluted with Mueller-Hinton agar (MHB) to an initial concentration of 200 ⁇ g/mL.
  • CCSI Clinical and Laboratory Standards Institute
  • Fresh rabbit red blood cells (RBCs) were centrifuged at 2500 rpm for 3 min and then washed twice with PBS. Rabbit red blood cells were then resuspended in PBS to prepare an 8% (v/v) cell suspension. Samples were first dissolved in DMSO or PBS (final concentration of DMSO ⁇ 0.5%) and then diluted with PBS to prepare two-fold gradient dilutions (400 to 3.125 ⁇ g/mL). Rabbit red blood cell suspension (100 ⁇ L) was mixed with a two-fold gradient dilution of the sample (100 ⁇ L) and added to a sterile 96-well plate.
  • the 96-well plate was incubated at 37°C for 1 hour and centrifuged at 2500 rpm for 5 minutes. Transfer the supernatant (100 ⁇ L) to a new 96-well plate, and use a multifunctional microplate reader to measure the absorbance at 576 nm.
  • the 2% Triton X-100 solution treatment group is used as a positive control, and the PBS or 0.5% DMSO treatment group is used. Used as negative control.
  • the initial MIC values of compound FT09 and norfloxacin against Staphylococcus aureus ATCC29213 and the initial MIC values of FT09 and amoxicillin against E. coli ATCC25922 were obtained by the above MIC determination method. Then take out the bacterial cells in the 96-well plate with a concentration of 0.5 ⁇ MIC to prepare a bacterial suspension (about 1 ⁇ 10 6 CFU/mL) for the next step of MIC value determination. After the sample was incubated at 37°C for 24 hours, the change in MIC value of the sample was measured. The experiment was carried out continuously for 22-23 days.
  • NCTC clone 929 The cytotoxicity of chlorphenol and its derivative FT09 on mouse fibroblasts was evaluated by CCK-8 method.
  • Log-phase mouse fibroblasts (NCTC clone 929) cells were digested with 0.25% trypsin (containing EDTA) for 3 minutes, then pipetted and diluted with RPMI 1640 medium (containing 10% fetal bovine serum FBS) and inoculated into 96 wells. plate (approximately 2 ⁇ 10 4 cells/well), incubate at 37°C for 24 h under a 5% CO 2 atmosphere and then remove the culture medium.
  • RPMI 1640 medium containing 10% fetal bovine serum FBS
  • Chlorphenol and its derivative FT09 were first dissolved in DMSO (final concentration of DMSO ⁇ 0.1%), and then diluted with RPMI 1640 medium (containing 10% fetal bovine serum FBS) to prepare a two-fold gradient dilution (200 to 3.125 ⁇ g/mL). Two-fold gradient dilutions (100 ⁇ L) of the above samples were added to a 96-well plate and incubated with NCTC clone 929 cells for 24 h; finally, a CCK-8 solution with a final concentration of 10 ⁇ L was added to each well and incubated in the incubator for 1.0 h. Then use a microplate reader to measure the absorbance of the solution at 450nm (OD 450 ).
  • mice Female C57BL6 mice (6-8 weeks, average weight 20g) were used in this study.
  • an immunosuppressed mouse model was established for the mouse corneal infection model induced by Staphylococcus aureus ATCC29213, while the corneal infection model induced by Pseudomonas aeruginosa ATCC9027 did not require immunosuppression of the mice.
  • mice Five days before infection, mice were injected intraperitoneally with cyclophosphamide (100 mg/kg) three times.
  • mice Bacterial cells (Staphylococcus aureus ATCC 29213) seeded on Mueller Hinton agar (MHA) plates were suspended with PBS, and the bacterial suspension concentration was adjusted to approximately 5 ⁇ 10 7 CFU/mL for corneal infection.
  • the mice were anesthetized by intraperitoneal injection of 2.5% Avertin (500 mg/kg) anesthetic, and then the cornea of the left eye of the mouse was scratched with a sterile needle, and 15 ⁇ L of bacterial suspension was dropped onto the damaged cornea.
  • mice were randomly divided into three groups (5 mice per group) and compounds (0.5% FT09, 5% glucose solution, 5% vancomycin, or 0.3% gatifloxacin) were topically applied four times daily.
  • mice administered continuously for 3 days. Finally, the mice were euthanized, the injured corneas were collected, and viable bacteria were counted by MHA plate counting. SPSS 22.0 software was used to calculate the P value, and P ⁇ 0.05 was considered to be statistically significant.
  • Compound FT09 shows excellent in vitro antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria, and has very high membrane selectivity and high safety. We further evaluate its use in animals. antibacterial effect.
  • mice were first treated with cyclophosphamide to immunosuppress the mice, and then a mouse corneal infection model induced by Staphylococcus aureus ATCC29213 was established; secondly, a mouse cornea infection model was directly induced by Pseudomonas aeruginosa ATCC9027. Infection model (mice were not immunosuppressed).
  • compound FT09 showed excellent in vivo antibacterial activity against Staphylococcus aureus ATCC29213 (positive bacteria) and Pseudomonas aeruginosa ATCC9027 (negative bacteria), which can reduce the amount of bacteria in the infected cornea. More than 99.9%, its efficacy is comparable to commercially available vancomycin or gatifloxacin. These results indicate that compound FT09 can cure mouse corneal infections caused by Staphylococcus aureus and Pseudomonas aeruginosa.

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Abstract

The present invention provides a clofoctol derivative, which has a structure represented by general formula (1). The clofoctol derivative has high efficiency, low toxicity, wide antibacterial spectrum, fast sterilization speed, excellent in-vivo antibacterial activity, and good druggability, and is capable of effectively avoiding the generation of bacterial drug resistance.

Description

氯福克酚衍生物、抗菌药物及制备方法与应用Chlorphenol derivatives, antibacterial drugs, preparation methods and applications 技术领域Technical field
本发明涉及抗菌领域，特别是涉及一种氯福克酚衍生物、抗菌药物及制备方法与应用。The present invention relates to the field of antibacterial, in particular to a chlorphenol derivative, an antibacterial drug, a preparation method and application.
背景技术Background technique
近年来，由于对抗菌药物的滥用和误用，导致抗菌药物的耐药性急剧增加，基本上所有的抗菌药物都已出现细菌耐药现象，已对21世纪的全球公共卫生安全构成了严重威胁。抗菌药物的耐药性一般会延长病人的住院时间并且增加医疗费用，让越来越多的严重感染病，如结核病、脑膜炎、肺炎和败血症等变得更加难以治疗，甚至面临无药可治的局面，导致死亡率急剧上升，给全球医疗体系带来严重的负担。据估计，目前每年约有70万名患者因耐药性细菌感染而死亡。若抗菌药物耐药性问题不得到有效控制，预计到2050年，每年将有1000万患者死于耐药细菌感染。20世纪80年代以来，新型抗菌药物的发现和开发进展十分缓慢，新批准的抗菌药物数量急剧减少。此外，在过去的三十年里，还未有新型抗菌药物被批准用于治疗革兰氏阴性菌感染。革兰氏阴性菌具有由脂多糖层和膜磷脂组成的特殊外膜，使得大多数抗菌药物难以穿透细菌细胞外膜以发挥有效的抗菌活性。因此，开发一种能够克服或者减缓耐药性产生的新型抗菌药物，特别是抗革兰氏阴性菌药物就显得极其重要。In recent years, the abuse and misuse of antibacterial drugs has led to a sharp increase in antibacterial drug resistance. Basically all antibacterial drugs have become resistant to bacteria, posing a serious threat to global public health security in the 21st century. . Antimicrobial resistance generally prolongs patients' hospitalization and increases medical costs, making more and more serious infections, such as tuberculosis, meningitis, pneumonia and sepsis, more difficult to treat or even untreatable. situation, leading to a sharp increase in mortality and placing a serious burden on the global medical system. It is estimated that approximately 700,000 patients currently die each year due to drug-resistant bacterial infections. If the problem of antimicrobial resistance is not effectively controlled, it is estimated that 10 million patients will die from drug-resistant bacterial infections every year by 2050. Since the 1980s, the discovery and development of new antibacterial drugs has progressed very slowly, and the number of newly approved antibacterial drugs has decreased sharply. Furthermore, no new antimicrobial drugs have been approved for the treatment of Gram-negative bacterial infections in the past three decades. Gram-negative bacteria have a special outer membrane composed of a lipopolysaccharide layer and membrane phospholipids, making it difficult for most antibacterial drugs to penetrate the bacterial cell outer membrane to exert effective antibacterial activity. Therefore, it is extremely important to develop new antibacterial drugs that can overcome or slow down the development of drug resistance, especially drugs against Gram-negative bacteria.
传统的大多数批准上市的抗菌药物仍然基于传统的抗菌分子骨架，容易导致交叉耐药，并且传统的抗菌药物的作用靶点单一，缺乏新型作用机制，容易产生耐药性。Most of the traditional antibacterial drugs approved for marketing are still based on traditional antibacterial molecular skeletons, which can easily lead to cross-resistance. Moreover, traditional antibacterial drugs have a single target and lack new mechanisms of action, which can easily lead to drug resistance.
发明内容Contents of the invention
基于此，本发明提供了一种氯福克酚衍生物，其高效低毒、抗菌谱广、杀菌速度快、体内抗菌活性优良、成药性好，并且能够有效避免细菌耐药性产生。Based on this, the present invention provides a chlorphenol derivative, which is highly efficient and low-toxic, has a broad antibacterial spectrum, fast sterilization speed, excellent antibacterial activity in vivo, good medicinal properties, and can effectively avoid the development of bacterial resistance.
本发明通过如下技术方案实现。The present invention is realized through the following technical solutions.
一种氯福克酚衍生物，其结构如通式(1)所示：A kind of chlorphenol derivative, its structure is shown as general formula (1):
Figure PCTCN2022088513-appb-000001
Figure PCTCN2022088513-appb-000001
其中：in:
L 1选自具有1至10个C原子的直链烷基或具有3至10个C原子的支链或环状的烷基； L 1 is selected from a linear alkyl group having 1 to 10 C atoms or a branched or cyclic alkyl group having 3 to 10 C atoms;
R 1选自胍基、氨基、精氨酸基团、组氨酸基团、赖氨酸基团或这些基团的组合。 R1 is selected from a guanidine group, an amino group, an arginine group, a histidine group, a lysine group or a combination of these groups.
在其中一个实施例中，L 1选自具有1至6个C原子的直链烷基或具有3至6个C原子的支链或环状的烷基。 In one embodiment, L 1 is selected from linear alkyl groups having 1 to 6 C atoms or branched or cyclic alkyl groups having 3 to 6 C atoms.
在其中一个实施例中，L 1选自具有1至4个C原子的直链烷基。 In one of the embodiments, L 1 is selected from linear alkyl groups having 1 to 4 C atoms.
在其中一个实施例中，其结构如通式(2-1)或(2-2)所示：In one embodiment, its structure is shown in general formula (2-1) or (2-2):
Figure PCTCN2022088513-appb-000002
Figure PCTCN2022088513-appb-000002
在其中一个实施例中，R 1选自如下基团的任一种： In one embodiment, R 1 is selected from any one of the following groups:
Figure PCTCN2022088513-appb-000003
Figure PCTCN2022088513-appb-000003
在其中一个实施例中，所述氯福克酚衍生物选自如下结构：In one embodiment, the chlorphenol derivative is selected from the following structures:
Figure PCTCN2022088513-appb-000004
Figure PCTCN2022088513-appb-000004
本发明还提供一种如上所述的氯福克酚衍生物的制备方法，包括如下步骤：The present invention also provides a method for preparing the chlorphenol derivative as described above, which includes the following steps:
将化合物
Figure PCTCN2022088513-appb-000005
进行威廉姆逊合成反应，制备中间体A
Figure PCTCN2022088513-appb-000006
将中间体A进行水解反应，制备中间体B
Figure PCTCN2022088513-appb-000007
compound
Figure PCTCN2022088513-appb-000005
Carry out Williamson synthesis reaction to prepare intermediate A
Figure PCTCN2022088513-appb-000006
Perform hydrolysis reaction of intermediate A to prepare intermediate B
Figure PCTCN2022088513-appb-000007
将所述中间体B进行脱水缩合反应，制备所述氯福克酚衍生物。The intermediate B is subjected to a dehydration condensation reaction to prepare the chlorphenol derivative.
在其中一个实施例中，威廉姆逊合成反应的温度为60℃～70℃，时间为4h～5h。In one of the embodiments, the temperature of the Williamson synthesis reaction is 60°C to 70°C, and the time is 4h to 5h.
本发明还提供如上所述的氯福克酚衍生物在制备抗菌药物中的应用。The present invention also provides the use of the above-mentioned chlorphenol derivatives in the preparation of antibacterial drugs.
本发明还提供一种抗菌药物，其组分包括如上所述的氯福克酚衍生物、盐以及药学上可接受的辅料。The present invention also provides an antibacterial drug, the components of which include the above-mentioned chlorphenol derivatives, salts and pharmaceutically acceptable excipients.
与现有技术相比较，本发明的氯福克酚衍生物具有如下有益效果：Compared with the prior art, the chlorphenol derivative of the present invention has the following beneficial effects:
本发明所述的氯福克酚衍生物以氯福克酚为分子骨架，具有两亲性的阳离子型化合物，具备新型的膜靶向抗菌机制，拓宽了氯福克酚的抗菌谱。本发明所述的氯福克酚衍生物对革兰氏阳性菌，包括耐甲氧西林金黄色葡萄球菌，以及革兰氏阴性菌，包括铜绿假单胞菌、大肠杆菌、鲍曼不动杆菌和肺炎克雷伯菌均表现出优异的体外和体内抗菌活性，其具有良好的水溶性和优良的体内药代动力学特性，对哺乳动物细胞表现出较低的细胞毒性和溶血活性，膜选择性高，具有较好的成药性。这类抗菌药物不仅具有较强的广谱抗菌活性，而且在实验室模拟的耐药性研究中能够克服细菌耐药性的产生。The clofol derivative of the present invention uses clofol as a molecular skeleton, is an amphipathic cationic compound, has a novel membrane-targeted antibacterial mechanism, and broadens the antibacterial spectrum of clofol. The chlorphenol derivatives of the present invention are effective against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, and Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii. and Klebsiella pneumoniae exhibit excellent in vitro and in vivo antibacterial activity, have good water solubility and excellent in vivo pharmacokinetic properties, and exhibit low cytotoxicity and hemolytic activity against mammalian cells. Membrane selection It has high potency and good medicinal properties. This type of antibacterial drugs not only have strong broad-spectrum antibacterial activity, but can also overcome the development of bacterial resistance in laboratory simulated drug resistance studies.
附图说明Description of the drawings
图1为本发明提供的耐药性研究；其中，A代表FT09和诺氟沙星对金黄色葡萄球菌ATCC29213的耐药性研究，B代表FT09和阿莫西林对大肠杆菌ATCC25922的耐药性研究；Figure 1 shows the drug resistance research provided by the present invention; wherein, A represents the research on the resistance of FT09 and norfloxacin to Staphylococcus aureus ATCC29213, and B represents the research on the resistance of FT09 and amoxicillin to Escherichia coli ATCC25922. ;
图2为本发明提供的细胞毒性研究；其中，A代表氯福克酚对小鼠成纤维细胞的细胞毒性，B代表FT09对小鼠成纤维细胞的细胞毒性；Figure 2 is a cytotoxicity study provided by the present invention; where A represents the cytotoxicity of chlorphenol on mouse fibroblasts, and B represents the cytotoxicity of FT09 on mouse fibroblasts;
图3为本发明提供的化合物FT09对金黄色葡萄球菌ATCC29213细胞膜完整性的影响；Figure 3 shows the effect of compound FT09 provided by the present invention on the integrity of the cell membrane of Staphylococcus aureus ATCC29213;
图4为本发明提供的体内抗菌活性研究；其中，A代表FT09和万古霉素对金黄色葡萄球菌ATCC29213的体内抗菌活性研究，B代表FT09和加替沙星对 铜绿假单胞菌ATCC9027的体内抗菌活性研究。Figure 4 is an in vivo antibacterial activity study provided by the present invention; wherein, A represents the in vivo antibacterial activity study of FT09 and vancomycin against Staphylococcus aureus ATCC29213, and B represents the in vivo antibacterial activity study of FT09 and gatifloxacin against Pseudomonas aeruginosa ATCC9027. Antibacterial activity studies.
具体实施方式Detailed ways
为了便于理解本发明，下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施方式。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施方式。相反地，提供这些实施方式的目的是使对本发明的公开内容的理解更加透彻全面。In order to facilitate understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Preferred embodiments of the invention are shown in the accompanying drawings. However, the invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough understanding of the present disclosure will be provided.
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施方式的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the invention belongs. The terminology used herein in the description of the invention is for the purpose of describing specific embodiments only and is not intended to limit the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
本发明所述基团和化合物中所涉及的元素碳、氢、氧、硫、氮或卤素均包括它们的同位素情况，及本发明所述基团和化合物中所涉及的元素碳、氢、氧、硫或氮任选进一步被一个或多个它们对应的同位素所替代。The elements carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds described in the present invention all include their isotope conditions, and the elements carbon, hydrogen, oxygen involved in the groups and compounds described in the present invention , sulfur or nitrogen are optionally further replaced by one or more of their corresponding isotopes.
术语“烷基”是指包含伯(正)碳原子、或仲碳原子、或叔碳原子、或季碳原子、或其组合的饱和烃。包含该术语的短语，例如，“C 1～C 9烷基”是指包含1～9个碳原子的烷基，每次出现时，可以互相独立地为C 1烷基、C 2烷基、C 3烷基、C 4烷基、C 5烷基、C 6烷基、C 7烷基、C 8烷基、C 9烷基。合适的实例包括但不限于：甲基(Me、-CH 3)、乙基(Et、-CH 2CH 3)、1-丙基(n-Pr、n-丙基、-CH 2CH 2CH 3)、2-丙基(i-Pr、i-丙基、-CH(CH 3) 2)、1-丁基(n-Bu、n-丁基、-CH 2CH 2CH 2CH 3)、2-甲基-1-丙基(i-Bu、i-丁基、-CH 2CH(CH 3) 2)、2-丁基(s-Bu、s-丁基、-CH(CH 3)CH 2CH 3)、2-甲基-2-丙基(t-Bu、t-丁基、-C(CH 3) 3)、1-戊基(n-戊基、-CH 2CH 2CH 2CH 2CH 3)、2-戊基(-CH(CH 3)CH 2CH 2CH 3)、3-戊基(-CH(CH 2CH 3) 2)、 2-甲基-2-丁基(-C(CH 3) 2CH 2CH 3)、3-甲基-2-丁基(-CH(CH 3)CH(CH 3) 2)、3-甲基-1-丁基(-CH 2CH 2CH(CH 3) 2)、2-甲基-1-丁基(-CH 2CH(CH 3)CH 2CH 3)、1-己基(-CH 2CH 2CH 2CH 2CH 2CH 3)、2-己基(-CH(CH 3)CH 2CH 2CH 2CH 3)、3-己基(-CH(CH 2CH 3)(CH 2CH 2CH 3))、2-甲基-2-戊基(-C(CH 3) 2CH 2CH 2CH 3)、3-甲基-2-戊基(-CH(CH 3)CH(CH 3)CH 2CH 3)、4-甲基-2-戊基(-CH(CH 3)CH 2CH(CH 3) 2)、3-甲基-3-戊基(-C(CH 3)(CH 2CH 3) 2)、2-甲基-3-戊基(-CH(CH 2CH 3)CH(CH 3) 2)、2,3-二甲基-2-丁基(-C(CH 3) 2CH(CH 3) 2)、3,3-二甲基-2-丁基(-CH(CH 3)C(CH 3) 3和辛基(-(CH 2) 7CH 3)。 The term "alkyl" refers to a saturated hydrocarbon containing primary (normal) carbon atoms, or secondary carbon atoms, or tertiary carbon atoms, or quaternary carbon atoms, or combinations thereof. Phrases containing this term, for example, "C 1 to C 9 alkyl" refer to alkyl groups containing 1 to 9 carbon atoms, and each occurrence may be independently C 1 alkyl, C 2 alkyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, C 6 alkyl, C 7 alkyl, C 8 alkyl, C 9 alkyl. Suitable examples include, but are not limited to: methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ) , 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2 CH (CH 3 ) 2 ), 2-butyl (s-Bu, s-butyl, -CH (CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-methyl-2- Butyl (-C(CH 3 ) 2 CH 2 CH 3 ), 3-methyl-2-butyl (-CH(CH 3 )CH(CH 3 ) 2 ), 3-methyl-1-butyl ( -CH 2 CH 2 CH(CH 3 ) 2 ), 2-methyl-1-butyl (-CH 2 CH(CH 3 )CH 2 CH 3 ), 1-hexyl (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-hexyl (-CH(CH 3 )CH 2 CH 2 CH 2 CH 3 ), 3-hexyl (-CH(CH 2 CH 3 )(CH 2 CH 2 CH 3 )), 2- Methyl-2-pentyl (-C(CH 3 ) 2 CH 2 CH 2 CH 3 ), 3-methyl-2-pentyl (-CH(CH 3 )CH(CH 3 )CH 2 CH 3 ), 4-Methyl-2-pentyl(-CH(CH 3 )CH 2 CH(CH 3 ) 2 ), 3-methyl-3-pentyl(-C(CH 3 )(CH 2 CH 3 ) 2 ) , 2-methyl-3-pentyl (-CH(CH 2 CH 3 )CH(CH 3 ) 2 ), 2,3-dimethyl-2-butyl (-C(CH 3 ) 2 CH(CH 3 ) 2 ), 3,3-dimethyl-2-butyl (-CH(CH 3 )C(CH 3 ) 3 and octyl (-(CH 2 ) 7 CH 3 ).
“氨基”是指氨的衍生物，具有式-N(X) 2的结构特征，其中每个“X”独立地是H、取代的或未被取代的烷基、取代的或未被取代的环烷基、取代的或未被取代的杂环基等。氨基的非限制性类型包括-NH 2、-N(烷基) 2、-NH(烷基)、-N(环烷基) 2、-NH(环烷基)、-N(杂环基) 2、-NH(杂环基)、-N(芳基) 2、-NH(芳基)、-N(烷基)(芳基)、-N(烷基)(杂环基)、-N(环烷基)(杂环基)、-N(芳基)(杂芳基)、-N(烷基)(杂芳基)等。 "Amino" refers to a derivative of ammonia having the structural characteristics of the formula -N(X) 2 , where each "X" is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted Cycloalkyl, substituted or unsubstituted heterocyclyl, etc. Non-limiting types of amino groups include -NH 2 , -N(alkyl) 2 , -NH(alkyl), -N(cycloalkyl) 2 , -NH(cycloalkyl), -N(heterocyclyl) 2 , -NH (heterocyclyl), -N (aryl) 2 , -NH (aryl), -N (alkyl) (aryl), -N (alkyl) (heterocyclyl), -N (Cycloalkyl)(heterocyclyl), -N(aryl)(heteroaryl), -N(alkyl)(heteroaryl), etc.
本发明提供了一种氯福克酚衍生物，其结构如通式(1)所示：The invention provides a chlorphenol derivative, the structure of which is shown in the general formula (1):
Figure PCTCN2022088513-appb-000008
Figure PCTCN2022088513-appb-000008
其中：in:
L 1选自具有1至10个C原子的直链烷基或具有3至10个C原子的支链或环状的烷基； L 1 is selected from a linear alkyl group having 1 to 10 C atoms or a branched or cyclic alkyl group having 3 to 10 C atoms;
R 1选自胍基、氨基、精氨酸基团、组氨酸基团、赖氨酸基团或这些基团的 组合。 R1 is selected from a guanidine group, an amino group, an arginine group, a histidine group, a lysine group or a combination of these groups.
在一个具体的示例中，L 1选自具有1至6个C原子的直链烷基或具有3至6个C原子的支链或环状的烷基。更具体地，L 1选自具有1至4个C原子的直链烷基。 In a specific example, L 1 is selected from linear alkyl groups having 1 to 6 C atoms or branched or cyclic alkyl groups having 3 to 6 C atoms. More specifically, L 1 is selected from linear alkyl groups having 1 to 4 C atoms.
在一个具体的示例中，其结构如通式(2-1)或(2-2)所示：In a specific example, its structure is as shown in general formula (2-1) or (2-2):
Figure PCTCN2022088513-appb-000009
Figure PCTCN2022088513-appb-000009
在一个具体的示例中，R 1选自如下基团的任一种： In a specific example, R 1 is selected from any one of the following groups:
Figure PCTCN2022088513-appb-000010
Figure PCTCN2022088513-appb-000010
在一个具体的示例中，氯福克酚衍生物选自如下结构：In a specific example, the chlorphenol derivative is selected from the following structures:
Figure PCTCN2022088513-appb-000011
Figure PCTCN2022088513-appb-000011
在一个具体的示例中，氯福克酚衍生物选自如下结构：In a specific example, the chlorphenol derivative is selected from the following structures:
Figure PCTCN2022088513-appb-000012
Figure PCTCN2022088513-appb-000012
可以理解地，氨基酸与氨基酸的连接位点默认为酰胺键。Understandably, the connection site between amino acids is the amide bond by default.
本发明还提供一种上述氯福克酚衍生物的制备方法，包括如下步骤：The present invention also provides a preparation method of the above-mentioned chlorphenol derivative, which includes the following steps:
将化合物
Figure PCTCN2022088513-appb-000013
进行威廉姆逊合成反应，制备中间体A
Figure PCTCN2022088513-appb-000014
将中间体A进行水解反应，制备中间体B
Figure PCTCN2022088513-appb-000015
compound
Figure PCTCN2022088513-appb-000013
Carry out Williamson synthesis reaction to prepare intermediate A
Figure PCTCN2022088513-appb-000014
Perform hydrolysis reaction of intermediate A to prepare intermediate B
Figure PCTCN2022088513-appb-000015
将中间体B进行脱水缩合反应，制备氯福克酚衍生物。The intermediate B is subjected to a dehydration condensation reaction to prepare a chlorphenol derivative.
在一个具体的示例中，威廉姆逊合成反应的温度为60℃～70℃，时间为4h～5h。In a specific example, the temperature of the Williamson synthesis reaction is 60°C to 70°C, and the time is 4h to 5h.
本发明还提供上述氯福克酚衍生物在制备抗菌药物中的应用。The present invention also provides the use of the above-mentioned chlorphenol derivatives in the preparation of antibacterial drugs.
本发明还提供一种抗菌药物，其组分包括上述氯福克酚衍生物、盐以及药学上可接受的辅料。The present invention also provides an antibacterial drug, the components of which include the above-mentioned chlorphenol derivatives, salts and pharmaceutically acceptable excipients.
以下结合具体实施例对本发明的氯福克酚衍生物及其制备方法做进一步详细的说明。以下实施例中所用的原料，如无特别说明，均为市售产品。The chlorphenol derivative of the present invention and its preparation method will be further described in detail below with reference to specific examples. The raw materials used in the following examples are all commercially available products unless otherwise specified.
实施例1Example 1
本实施例提供一种氯福克酚衍生物FT07及其制备方法，合成路线如下：This embodiment provides a chlorphenol derivative FT07 and its preparation method. The synthesis route is as follows:
Figure PCTCN2022088513-appb-000016
Figure PCTCN2022088513-appb-000016
中间体FT05：
Figure PCTCN2022088513-appb-000017
Intermediate FT05:
Figure PCTCN2022088513-appb-000017
将氯福克酚(400.0mg，1.09mmol)溶解在15mL丙酮中，同时加入碳酸钾(302.6mg，2.19mmol)和溴乙酸乙酯(293μL，2.74mmol)。混合均匀后将反应液加热至65℃搅拌回流4h。然后反应液用乙酸乙酯萃取两次后浓缩有机相，使用柱层析分离纯化。经干燥后得到白色固体产物FT05(438.6mg，89％)。 1H NMR(400MHz,CDCl 3)δ7.37(d,J＝0.9Hz,1H),7.19–7.12(m,2H),7.10(d,J＝1.1Hz,2H),6.66(d,J＝8.5Hz,1H),4.58(s,2H),4.24(q,J＝7.1Hz,2H),4.09(s,2H),1.65(s,2H),1.31–1.26(m,9H),0.67(s,9H). 13C NMR(100MHz,CDCl 3)δ169.12,153.64,143.21,137.38,134.84,132.12,131.80,129.36,128.89,126.82,126.72,125.29,110.77,65.76,61.27,56.97,38.00,33.52,32.33,31.79,31.66,14.19.HRMS(ESI+):calculated for C 25H 32Cl 2O 3[M+Na] +473.1621,found 473.1620. Chlorphenol (400.0 mg, 1.09 mmol) was dissolved in 15 mL acetone, and potassium carbonate (302.6 mg, 2.19 mmol) and ethyl bromoacetate (293 μL, 2.74 mmol) were added simultaneously. After mixing evenly, the reaction solution was heated to 65°C, stirred and refluxed for 4 hours. The reaction solution was extracted twice with ethyl acetate, the organic phase was concentrated, and column chromatography was used for separation and purification. After drying, the product FT05 was obtained as a white solid (438.6 mg, 89%). 1 H NMR (400MHz, CDCl 3 ) δ7.37 (d, J＝0.9Hz, 1H), 7.19–7.12 (m, 2H), 7.10 (d, J＝1.1Hz, 2H), 6.66 (d, J＝ 8.5Hz,1H),4.58(s,2H),4.24(q,J＝7.1Hz,2H),4.09(s,2H),1.65(s,2H),1.31–1.26(m,9H),0.67( s,9H). 13 C NMR (100MHz, CDCl 3 ) δ169.12,153.64,143.21,137.38,134.84,132.12,131.80,129.36,128.89,126.82,126.72,125.29,110.77,65.76, 61.27,56.97,38.00,33.52, 32.33,31.79,31.66,14.19.HRMS(ESI+):calculated for C 25 H 32 Cl 2 O 3 [M+Na] + 473.1621, found 473.1620.
产物FT07：
Figure PCTCN2022088513-appb-000018
Product FT07:
Figure PCTCN2022088513-appb-000018
将FT05(85.3mg，0.20mmol)用4mL四氢呋喃溶解后，加入2mL氢氧化锂水溶液(29.0mg，1.20mmol)。混合均匀后于常温条件下搅拌反应1.5小时。反应完全后加入冰乙酸调节至中性。反应液用正丁醇萃取后浓缩有机相，真空干燥后所得中间体用DMF(8mL)溶解，并依次加入2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU，230.4mg，0.60mmol)、N,N-二异 丙基乙胺(DIPEA，351μL，2.01mmol)和L-精氨酸甲酯二盐酸盐(157.3mg，0.60mmol)。混合均匀后，反应液在室温条件下搅拌反应24小时。反应完全后，反应液用正丁醇萃取两次后浓缩有机相，使用半制备型高效液相分离纯化。经干燥后得到黄色固体产物FT07(70.2mg,59％)。 1H NMR(400MHz,CD 3OD)δ7.46(d,J＝2.1Hz,1H),7.28–7.19(m,2H),7.15–7.09(m,2H),6.87(d,J＝8.6Hz,1H),4.59–4.50(m,3H),4.14(s,2H),3.73(s,3H),3.23–3.13(m,2H),2.00–1.65(m,4H),1.62–1.52(m,2H),1.29(s,6H),0.67(s,9H). 13C NMR(100MHz,CD 3OD)δ171.26,169.65,158.67,154.73,144.55,138.74,135.95,133.58,132.93,130.17,129.98,128.22,127.76,126.71,112.61,68.49,57.83,53.03,52.68,41.77,38.90,34.30,33.08,32.30,32.27,29.81,26.10.HRMS(ESI+):calculated for C 30H 42Cl 2N 4O 4[M+H] +593.2656,found 593.2666. After dissolving FT05 (85.3 mg, 0.20 mmol) in 4 mL of tetrahydrofuran, 2 mL of lithium hydroxide aqueous solution (29.0 mg, 1.20 mmol) was added. After mixing evenly, stir and react at room temperature for 1.5 hours. After the reaction is complete, add glacial acetic acid to adjust to neutrality. The reaction solution was extracted with n-butanol, the organic phase was concentrated, and the intermediate obtained after vacuum drying was dissolved in DMF (8 mL), and 2-(7-azebenzotriazole)-N,N,N',N was added in sequence. '-Tetramethylurea hexafluorophosphate (HATU, 230.4 mg, 0.60 mmol), N,N-diisopropylethylamine (DIPEA, 351 μL, 2.01 mmol) and L-arginine methyl ester dihydrochloride (157.3mg, 0.60mmol). After mixing evenly, the reaction solution was stirred and reacted at room temperature for 24 hours. After the reaction is complete, the reaction solution is extracted twice with n-butanol, the organic phase is concentrated, and semi-preparative high-performance liquid phase separation and purification are used. After drying, the product FT07 was obtained as a yellow solid (70.2 mg, 59%). 1 H NMR (400MHz, CD 3 OD) δ7.46 (d, J = 2.1Hz, 1H), 7.28–7.19 (m, 2H), 7.15–7.09 (m, 2H), 6.87 (d, J = 8.6Hz ,1H),4.59–4.50(m,3H),4.14(s,2H),3.73(s,3H),3.23–3.13(m,2H),2.00–1.65(m,4H),1.62–1.52(m ,2H),1.29(s,6H),0.67(s,9H). 13 C NMR(100MHz,CD 3 OD)δ171.26,169.65,158.67,154.73,144.55,138.74,135.95,133.58,132.93,130.17,129.9 8, 128.22,127.76,126.71,112.61,68.49,57.83,53.03,52.68,41.77,38.90,34.30,33.08,32.30,32.27,29.81,26.10.HRMS(ESI+):calculated for C 30 H 42 Cl 2 N 4 O 4 [ M+H] + 593.2656, found 593.2666.
实施例2Example 2
本实施例提供一种氯福克酚衍生物FT09及其制备方法，合成路线如下：This embodiment provides a chlorphenol derivative FT09 and its preparation method. The synthesis route is as follows:
Figure PCTCN2022088513-appb-000019
Figure PCTCN2022088513-appb-000019
产物T09：
Figure PCTCN2022088513-appb-000020
Product T09:
Figure PCTCN2022088513-appb-000020
将反应物FT05(80.0mg，0.18mmol)、HATU(168.9mg，0.44mmol)、DIPEA(154μL，0.88mmol)和H-Arg-Arg-Arg-OMe·4HCl(129.0mg，0.20mmol)按照合成FT07的方法，制备得到白色固体产物FT09(138.2mg，88％)。 1H NMR(400MHz,CD 3OD)δ7.45(d,J＝2.1Hz,1H),7.27–7.20(m,2H),7.13(d,J＝8.3Hz,1H),7.07(d,J＝2.3Hz,1H),6.86(d,J＝8.6Hz,1H),4.57(d,J＝5.2Hz,2H),4.49–4.36(m,3H),4.13(d,J＝3.1Hz,2H),3.72(s,3H),3.24–3.14(m,6H),1.94–1.59(m,14H),1.28(s,6H),0.65(s,9H). 13C NMR(100MHz,CD 3OD)δ174.04,173.60,173.56,171.28,170.54,158.70,154.75,144.49,138.71,136.00,133.60,133.10,129.99,129.96,128.22,127.79,126.68,112.66,68.54,57.82,54.22,53.87,53.37,52.87,41.91,41.86,41.81,38.89,34.16,33.06,32.29,32.26,30.38,30.12,29.42,26.23,26.15,25.95.HRMS(ESI+):calculated for C 42H 66Cl 2N 12O 6[M+2H] 2+453.2375,found 453.2380. The reactants FT05 (80.0 mg, 0.18 mmol), HATU (168.9 mg, 0.44 mmol), DIPEA (154 μL, 0.88 mmol) and H-Arg-Arg-Arg-OMe·4HCl (129.0 mg, 0.20 mmol) were synthesized according to the method of synthesizing FT07 Using this method, a white solid product FT09 (138.2 mg, 88%) was prepared. 1 H NMR (400MHz, CD 3 OD) δ7.45(d,J＝2.1Hz,1H),7.27–7.20(m,2H),7.13(d,J＝8.3Hz,1H),7.07(d,J ＝2.3Hz,1H),6.86(d,J＝8.6Hz,1H),4.57(d,J＝5.2Hz,2H),4.49–4.36(m,3H),4.13(d,J＝3.1Hz,2H ),3.72(s,3H),3.24–3.14(m,6H),1.94–1.59(m,14H),1.28(s,6H),0.65(s,9H). 13 C NMR(100MHz,CD 3 OD )δ174.04,173.60,173.56,171.28,170.54,158.70,154.75,144.49,138.71,136.00,133.60,133.10,129.99,129.96,128.22,127.79,126.68,1 12.66,68.54,57.82,54.22,53.87,53.37,52.87,41.91 ,41.86,41.81,38.89,34.16,33.06,32.29,32.26,30.38,30.12,29.42,26.23,26.15,25.95.HRMS(ESI+):calculated for C 42 H 66 Cl 2 N 12 O 6 [M+2H] 2 + 453.2375, found 453.2380.
实施例3Example 3
本实施例提供一种氯福克酚衍生物FT12及其制备方法，合成路线如下：This embodiment provides a chlorphenol derivative FT12 and its preparation method. The synthesis route is as follows:
Figure PCTCN2022088513-appb-000021
Figure PCTCN2022088513-appb-000021
产物FT12：
Figure PCTCN2022088513-appb-000022
Product FT12:
Figure PCTCN2022088513-appb-000022
将反应物FT07(64.4mg，0.11mmol)、HATU(120.9mg，0.33mmol)、DIPEA(111μL，0.65mmol)和H-Arg-Arg-NH 2·3HCl(89.4mg，0.20mmol)按照合成FT07的方法，制备得到黄色油状产物FT12(49.7mg，51％)。 1H NMR(400MHz,CD 3OD)δ7.46(d,J＝2.1Hz,1H),7.28–7.20(m,2H),7.13(d,J＝8.3Hz,1H),7.07(d,J＝2.4Hz,1H),6.87(d,J＝8.6Hz,1H),4.62–4.51(m,2H),4.50–4.43(m,1H),4.41–4.31(m,2H),4.19–4.08(m,2H),3.23–3.15(m,6H),1.96–1.59(m,14H),1.28(s,6H),0.66(s,9H).HRMS(ESI+):calculated for C 41H 65Cl 2N 13O 5[M+H] +890.4681,found 890.4685. The reactants FT07 (64.4 mg, 0.11 mmol), HATU (120.9 mg, 0.33 mmol), DIPEA (111 μL, 0.65 mmol) and H-Arg-Arg-NH 2 ·3HCl (89.4 mg, 0.20 mmol) were synthesized according to the method for synthesizing FT07 Method, the yellow oily product FT12 (49.7 mg, 51%) was prepared. 1 H NMR (400MHz, CD 3 OD) δ7.46(d,J＝2.1Hz,1H),7.28–7.20(m,2H),7.13(d,J＝8.3Hz,1H),7.07(d,J ＝2.4Hz,1H),6.87(d,J＝8.6Hz,1H),4.62–4.51(m,2H),4.50–4.43(m,1H),4.41–4.31(m,2H),4.19–4.08( m,2H),3.23–3.15(m,6H),1.96–1.59(m,14H),1.28(s,6H),0.66(s,9H).HRMS(ESI+):calculated for C 41 H 65 Cl 2 N 13 O 5 [M+H] + 890.4681,found 890.4685.
实施例4Example 4
本实施例提供一种氯福克酚衍生物FT37及其制备方法，合成路线如下：This embodiment provides a chlorphenol derivative FT37 and its preparation method. The synthesis route is as follows:
Figure PCTCN2022088513-appb-000023
Figure PCTCN2022088513-appb-000023
产物FT37：
Figure PCTCN2022088513-appb-000024
Product FT37:
Figure PCTCN2022088513-appb-000024
将反应物FT05(75mg，0.18mmol)、HATU(202.1mg，0.53mmol)、DIPEA(185μL，1.06mmol)和H-Arg-Arg-OMe·3HCl(116.4mg，0.27mmol)按照合成FT07的方法，制备得到黄白色固体产物FT37(103.6mg，80％)。 1H NMR(400MHz,CD 3OD)δ7.45(d,J＝2.1Hz,1H),7.27–7.17(m,2H),7.12(d,J＝8.3Hz,1H),7.08(d,J＝2.2Hz,1H),6.86(d,J＝8.6Hz,1H),4.55(d,J＝2.4Hz,2H),4.51–4.43(m,2H),4.18–4.07(m,2H),3.73(s,3H),3.24–3.10(m,4H),2.03–1.82(m,2H),1.79–1.53(m,8H),1.28(s,6H),0.66(s,9H). 13C NMR(100MHz,CD 3OD)δ173.59,173.55,171.05,170.37,158.71,154.71,144.51,138.69,136.01,133.59,133.02,130.05,129.98,128.22,127.77,126.68,112.60,68.49,57.83,53.63,53.25,52.90,41.91,41.78,38.89,34.21,33.07,32.29,32.26,32.24,30.58,29.44,26.22,25.91.HRMS(ESI+):calculated for C 36H 54Cl 2N 8O 5[M+H] +749.3667,found749.3671. The reactants FT05 (75mg, 0.18mmol), HATU (202.1mg, 0.53mmol), DIPEA (185μL, 1.06mmol) and H-Arg-Arg-OMe·3HCl (116.4mg, 0.27mmol) were synthesized according to the method of synthesizing FT07, The yellow-white solid product FT37 (103.6 mg, 80%) was prepared. 1 H NMR (400MHz, CD 3 OD) δ7.45(d,J＝2.1Hz,1H),7.27–7.17(m,2H),7.12(d,J＝8.3Hz,1H),7.08(d,J ＝2.2Hz,1H),6.86(d,J＝8.6Hz,1H),4.55(d,J＝2.4Hz,2H),4.51–4.43(m,2H),4.18–4.07(m,2H),3.73 (s,3H),3.24–3.10(m,4H),2.03–1.82(m,2H),1.79–1.53(m,8H),1.28(s,6H),0.66(s,9H). 13 C NMR (100MHz, CD 3 OD) δ173.59,173.55,171.05,170.37,158.71,154.71,144.51,138.69,136.01,133.59,133.02,130.05,129.98,128.22,127.77,126. 68,112.60,68.49,57.83,53.63,53.25,52.90 ,41.91,41.78,38.89,34.21,33.07,32.29,32.26,32.24,30.58,29.44,26.22,25.91.HRMS(ESI+):calculated for C 36 H 54 Cl 2 N 8 O 5 [M+H] + 749.3667, found749.3671.
实施例5Example 5
本实施例提供一种氯福克酚衍生物FT40及其制备方法，具体制备步骤如下：This embodiment provides a chlorphenol derivative FT40 and its preparation method. The specific preparation steps are as follows:
产物FT40：
Figure PCTCN2022088513-appb-000025
Product FT40:
Figure PCTCN2022088513-appb-000025
将反应物FT09(70.9mg，0.08mmol)用4mL四氢呋喃溶解后，加入2mL氢氧化锂水溶液(18.8mg，0.78mmol)。混合均匀后于常温条件下搅拌反应1.5小时。反应完全后加入冰乙酸调节至中性。反应液用正丁醇萃取后浓缩有机相，使用半制备型高效液相分离纯化。经干燥后得到淡黄色油状产物FT40(58.7mg，84％)。 1H NMR(400MHz,CD 3OD)δ7.45(d,J＝2.1Hz,1H),7.28–7.19(m,2H),7.12(d,J＝8.3Hz,1H),7.08(d,J＝2.1Hz,1H),6.86(d,J＝8.6Hz,1H),4.61–4.53(m,2H),4.52–4.46(m,1H),4.43–4.35(m,1H),4.23–4.16(m,1H),4.15–4.10(m,2H),3.25–3.11(m,6H),2.01–1.55(m,14H),1.28(s,6H),0.66(s,9H). 13C NMR(100MHz,CD 3OD)δ178.05,173.66,173.21,171.08,169.47,158.71,158.66,154.72,144.49,138.67,136.00,133.59,133.03,130.04,129.98,128.22,127.77,126.67,112.63,68.50,57.83,55.30,54.56,53.91,42.06,42.04,41.78,38.89,34.21,33.06,32.30,32.27,32.24,30.88,30.68,30.13,26.15,25.84.HRMS(ESI+):calculated for C 41H 64Cl 2N 12O 6[M+H] +891.4522,found 891.4517. After the reactant FT09 (70.9 mg, 0.08 mmol) was dissolved in 4 mL of tetrahydrofuran, 2 mL of lithium hydroxide aqueous solution (18.8 mg, 0.78 mmol) was added. After mixing evenly, stir and react at room temperature for 1.5 hours. After the reaction is complete, add glacial acetic acid to adjust to neutrality. The reaction solution was extracted with n-butanol, the organic phase was concentrated, and semi-preparative high-performance liquid phase separation was used for purification. After drying, the product FT40 (58.7 mg, 84%) was obtained as a light yellow oil. 1 H NMR (400MHz, CD 3 OD) δ7.45(d,J＝2.1Hz,1H),7.28–7.19(m,2H),7.12(d,J＝8.3Hz,1H),7.08(d,J ＝2.1Hz,1H),6.86(d,J＝8.6Hz,1H),4.61–4.53(m,2H),4.52–4.46(m,1H),4.43–4.35(m,1H),4.23–4.16( m,1H),4.15–4.10(m,2H),3.25–3.11(m,6H),2.01–1.55(m,14H),1.28(s,6H),0.66(s,9H). 13 C NMR( 100MHz, CD 3 OD) δ178.05,173.66,173.21,171.08,169.47,158.71,158.66,154.72,144.49,138.67,136.00,133.59,133.03,130.04,129.98,128. 22,127.77,126.67,112.63,68.50,57.83,55.30, 54.56,53.91,42.06,42.04,41.78,38.89,34.21,33.06,32.30,32.27,32.24,30.88,30.68,30.13,26.15,25.84.HRMS(ESI+):calculated for C 41 H 64 Cl 2 N 1 2 O 6 [ M+H] + 891.4522, found 891.4517.
实施例6Example 6
本实施例提供一种氯福克酚衍生物FT41及其制备方法，合成路线如下：This embodiment provides a chlorphenol derivative FT41 and its preparation method. The synthesis route is as follows:
Figure PCTCN2022088513-appb-000026
Figure PCTCN2022088513-appb-000026
产物FT41：
Figure PCTCN2022088513-appb-000027
Product FT41:
Figure PCTCN2022088513-appb-000027
将反应物FT07(50.3mg，0.09mmol)、HATU(99.0mg，0.26mmol)、DIPEA(91μL，0.52mmol)和H-Arg-Arg-Arg-OMe·4HCl(86.9mg，0.14mmol)按照合成FT07的方法，制备得到黄色油状产物FT41(38.6mg，42％)。 1H NMR(400MHz,CD 3OD)δ7.46(d,J＝2.0Hz,1H),7.27–7.20(m,2H),7.13(d,J＝8.3Hz,1H),7.07(d,J＝1.8Hz,1H),6.90–6.84(m,1H),4.63–4.51(m,2H),4.49–4.30(m,4H),4.18–4.07(m,2H),3.72(s,3H),3.24–3.13(m,8H),1.98–1.82(m,4H),1.80–1.57(m,14H),1.28(s,6H),0.65(s,9H). 13C NMR(100MHz,CD 3OD)δ174.11,173.90,173.72,173.58,171.31,170.12,158.68,154.72,144.42,138.68,135.98,133.57,133.09,129.97,129.95,128.21,127.75,126.64,112.62,101.32,68.52,57.79,54.37,54.28,53.93,53.38,52.88,41.89,41.83,41.79,41.73,38.86,34.16,33.05,32.30,32.26,30.29,30.16,29.95,29.39,26.29,26.17,26.13,25.96.HRMS(ESI+):calculated for C 48H 78Cl 2N 16O 7[M+2H] 2+531.2881,found 531.2896. The reactants FT07 (50.3mg, 0.09mmol), HATU (99.0mg, 0.26mmol), DIPEA (91μL, 0.52mmol) and H-Arg-Arg-Arg-OMe·4HCl (86.9mg, 0.14mmol) were synthesized according to the method of synthesizing FT07 Using this method, the yellow oily product FT41 (38.6 mg, 42%) was prepared. 1 H NMR (400MHz, CD 3 OD) δ7.46(d,J＝2.0Hz,1H),7.27–7.20(m,2H),7.13(d,J＝8.3Hz,1H),7.07(d,J ＝1.8Hz,1H),6.90–6.84(m,1H),4.63–4.51(m,2H),4.49–4.30(m,4H),4.18–4.07(m,2H),3.72(s,3H), 3.24–3.13(m,8H),1.98–1.82(m,4H),1.80–1.57(m,14H),1.28(s,6H),0.65(s,9H). 13 C NMR (100MHz, CD 3 OD )δ174.11,173.90,173.72,173.58,171.31,170.12,158.68,154.72,144.42,138.68,135.98,133.57,133.09,129.97,129.95,128.21,127.75,1 26.64,112.62,101.32,68.52,57.79,54.37,54.28,53.93 ,53.38,52.88,41.89,41.83,41.79,41.73,38.86,34.16,33.05,32.30,32.26,30.29,30.16,29.95,29.39,26.29,26.17,26.13,25.96.HRMS(ESI+ ):calculated for C 48 H 78 Cl 2 N 16 O 7 [M+2H] 2+ 531.2881,found 531.2896.
实施例7Example 7
本实施例提供一种氯福克酚衍生物FT42及其制备方法，合成路线如下：This embodiment provides a chlorphenol derivative FT42 and its preparation method. The synthesis route is as follows:
Figure PCTCN2022088513-appb-000028
Figure PCTCN2022088513-appb-000028
产物FT42：
Figure PCTCN2022088513-appb-000029
Product FT42:
Figure PCTCN2022088513-appb-000029
将反应物FT37(94.6mg，0.13mmol)、HATU(144.1mg，0.38mmol)、DIPEA(132μL，0.76mmol)和H-Arg-Arg-Arg-OMe·4HCl(126.5mg，0.20mmol)按照合成FT07的方法，制备得到黄色油状产物FT42(40.7mg，26％)。 1H NMR(400MHz,CD 3OD)δ7.46(d,J＝2.0Hz,1H),7.26–7.20(m,2H),7.13(d,J＝8.3Hz,1H),7.07(d,J＝1.9Hz,1H),6.87(d,J＝8.6Hz,1H),4.64–4.52(m,2H),4.49–4.28(m,5H),4.19–4.08(m,2H),3.73–3.71(m,3H),3.25–3.12(m,10H),1.98–1.55(m,22H),1.28(s,6H),0.66(s,9H). 13C NMR(100MHz,CD 3OD)δ174.16,174.04,173.94,173.82,173.60,171.41,169.98,158.77,158.74,158.72,158.70,154.76,144.50,138.71,136.01,133.62,133.09,129.98,128.23,127.79,126.67,112.65,101.34,77.10,68.61,57.82,54.34,54.13,53.41,52.88,52.39,41.92,41.88,41.86,41.83,41.81,38.89,34.17,33.07,32.32,32.29,32.26,32.21,32.19,29.42,27.29,26.31,26.20,26.13,26.03,20.85.HRMS(ESI+):calculated for C 54H 90Cl 2N 20O 8[M+2H] 2+609.3387,found 609.3397. The reactants FT37 (94.6mg, 0.13mmol), HATU (144.1mg, 0.38mmol), DIPEA (132μL, 0.76mmol) and H-Arg-Arg-Arg-OMe·4HCl (126.5mg, 0.20mmol) were synthesized according to the method of synthesizing FT07 Using this method, the yellow oily product FT42 (40.7 mg, 26%) was prepared. 1 H NMR (400MHz, CD 3 OD) δ7.46(d,J＝2.0Hz,1H),7.26–7.20(m,2H),7.13(d,J＝8.3Hz,1H),7.07(d,J ＝1.9Hz,1H),6.87(d,J＝8.6Hz,1H),4.64–4.52(m,2H),4.49–4.28(m,5H),4.19–4.08(m,2H),3.73–3.71( m,3H),3.25–3.12(m,10H),1.98–1.55(m,22H),1.28(s,6H),0.66(s,9H). 13 C NMR(100MHz,CD 3 OD)δ174.16,174.04 ,173.94,173.82,173.60,171.41,169.98,158.77,158.74,158.72,158.70,154.76,144.50,138.71,136.01,133.62,133.09,129.98,128.23,1 27.79,126.67,112.65,101.34,77.10,68.61,57.82,54.34 ,54.13,53.41,52.88,52.39,41.92,41.88,41.86,41.83,41.81,38.89,34.17,33.07,32.32,32.29,32.26,32.21,32.19,29.42,27.29,26.31, 26.20,26.13,26.03,20.85.HRMS (ESI+):calculated for C 54 H 90 Cl 2 N 20 O 8 [M+2H] 2+ 609.3387, found 609.3397.
实施例8Example 8
本实施例提供一种氯福克酚衍生物FT46及其制备方法，制备过程如下：This embodiment provides a chlorphenol derivative FT46 and its preparation method. The preparation process is as follows:
产物FT46：
Figure PCTCN2022088513-appb-000030
Product FT46:
Figure PCTCN2022088513-appb-000030
将反应物FT05(60mg，0.13mmol)用4mL四氢呋喃溶解后，加入2mL氢氧化锂水溶液(19.1mg，0.80mmol)。混合均匀后于常温条件下搅拌反应1.5小时。反应完全后加入冰乙酸调节至中性。反应液用正丁醇萃取后浓缩有机相。真空干燥后得到的中间体用DMF(8mL)溶解、同时加入HATU(152.4mg，0.40mmol)、DIPEA(232μL，1.33mmol)和N-Boc-L-赖氨酸甲酯盐酸盐(118.6mg，0.40mmol)。混合均匀后，反应液在室温条件下搅拌反应24小时。反应完全后，反应液用正丁醇萃取两次后浓缩有机相，干燥后用二氯甲烷(15mL)溶解，并加入三氟乙酸(3mL)，混合搅拌1小时后，用正丁醇萃取两次后浓缩有机相，使用半制备型高效液相分离纯化。制备得到黄色油状产物FT46(27.3mg，34％)。 1H NMR(400MHz,CD 3OD)δ7.46(d,J＝2.1Hz,1H),7.29–7.18(m,2H),7.14–7.06(m,2H),6.86(d,J＝8.6Hz,1H),4.60–4.46(m,3H),4.13(s,2H),3.72(s,3H),2.93–2.85(m,2H),1.97–1.60(m,6H),1.42–1.33(m,2H),1.30(s,6H),0.67(s,9H). 13C NMR(100MHz,CD 3OD)δ173.27,171.18,154.72,144.48,138.74,135.93,133.54,132.90,130.21,129.97,128.23,127.65,126.71,112.52,68.39,57.82,52.99,52.79,40.38,38.89,34.36,33.10,32.32,32.28,32.08,27.96,23.64.HRMS(ESI+):calculated for C 30H 42Cl 2N 2O 4[M+H] +565.2594,found565.2607. After the reactant FT05 (60 mg, 0.13 mmol) was dissolved in 4 mL of tetrahydrofuran, 2 mL of lithium hydroxide aqueous solution (19.1 mg, 0.80 mmol) was added. After mixing evenly, stir and react at room temperature for 1.5 hours. After the reaction is complete, add glacial acetic acid to adjust to neutrality. The reaction solution was extracted with n-butanol and the organic phase was concentrated. The intermediate obtained after vacuum drying was dissolved in DMF (8 mL), and HATU (152.4 mg, 0.40 mmol), DIPEA (232 μL, 1.33 mmol) and N-Boc-L-lysine methyl ester hydrochloride (118.6 mg) were added. ,0.40mmol). After mixing evenly, the reaction solution was stirred and reacted at room temperature for 24 hours. After the reaction is complete, the reaction solution is extracted twice with n-butanol, the organic phase is concentrated, dried, dissolved in dichloromethane (15 mL), and trifluoroacetic acid (3 mL) is added, mixed and stirred for 1 hour, and extracted twice with n-butanol. After several times, the organic phase was concentrated and purified using semi-preparative high-performance liquid phase separation. The product FT46 was prepared as a yellow oil (27.3 mg, 34%). 1 H NMR (400MHz, CD 3 OD) δ7.46 (d, J＝2.1Hz, 1H), 7.29–7.18 (m, 2H), 7.14–7.06 (m, 2H), 6.86 (d, J＝8.6Hz ,1H),4.60–4.46(m,3H),4.13(s,2H),3.72(s,3H),2.93–2.85(m,2H),1.97–1.60(m,6H),1.42–1.33(m ,2H),1.30(s,6H),0.67(s,9H). 13 C NMR(100MHz,CD 3 OD)δ173.27,171.18,154.72,144.48,138.74,135.93,133.54,132.90,130.21,129.97,128.2 3, 127.65,126.71,112.52,68.39,57.82,52.99,52.79,40.38,38.89,34.36,33.10,32.32,32.28,32.08,27.96,23.64.HRMS(ESI+):calculated for C 30 H 42 Cl 2 N 2 O 4 [ M+H] + 565.2594,found565.2607.
实施例9Example 9
本实施例提供一种氯福克酚衍生物FT49及其制备方法，合成路线如下：This embodiment provides a chlorphenol derivative FT49 and its preparation method. The synthesis route is as follows:
Figure PCTCN2022088513-appb-000031
Figure PCTCN2022088513-appb-000031
产物FT49：
Figure PCTCN2022088513-appb-000032
Product FT49:
Figure PCTCN2022088513-appb-000032
将反应物FT05(71.8mg，0.16mmol)、HATU(181.5mg，0.48mmol)、DIPEA(166μL，0.95mmol)和H-Arg-Arg-NH 2·3HCl(104.6mg，0.24mmol)按照合成FT07的方法，制备得到白色固体产物FT49(86.3mg，74％)。 1H NMR(400MHz,CD 3OD)δ7.45(d,J＝2.1Hz,1H),7.27–7.18(m,2H),7.13(d,J＝8.3Hz,1H),7.07(d,J＝2.3Hz,1H),6.87(d,J＝8.6Hz,1H),4.59–4.45(m,3H),4.42–4.36(m,1H),4.19–4.08(m,2H),3.18(t,J＝6.8Hz,4H),1.97–1.53(m,10H),1.28(s,6H),0.66(s,9H). 13C NMR(100MHz,CD 3OD)δ173.42,171.17,169.88,158.68,158.65,154.70,144.45,138.68,136.02,133.59,133.06,129.97,128.22,127.74,126.66,112.54,101.45,68.46,57.81,53.93,53.90,53.75,41.86,38.88,34.20,33.08,32.32,32.27,30.35,26.27,25.83.HRMS(ESI+):calculated for C 35H 53Cl 2N 9O 4[M+H] +734.3670,found 734.3659. The reactants FT05 (71.8 mg, 0.16 mmol), HATU (181.5 mg, 0.48 mmol), DIPEA (166 μL, 0.95 mmol) and H-Arg-Arg-NH 2 ·3HCl (104.6 mg, 0.24 mmol) were synthesized according to the method for synthesizing FT07 Method, a white solid product FT49 (86.3 mg, 74%) was prepared. 1 H NMR (400MHz, CD 3 OD) δ7.45(d,J＝2.1Hz,1H),7.27–7.18(m,2H),7.13(d,J＝8.3Hz,1H),7.07(d,J ＝2.3Hz,1H),6.87(d,J＝8.6Hz,1H),4.59–4.45(m,3H),4.42–4.36(m,1H),4.19–4.08(m,2H),3.18(t, J＝6.8Hz, 4H), 1.97–1.53 (m, 10H), 1.28 (s, 6H), 0.66 (s, 9H). 13 C NMR (100MHz, CD 3 OD) δ173.42, 171.17, 169.88, 158.68, 158.65 ,154.70,144.45,138.68,136.02,133.59,133.06,129.97,128.22,127.74,126.66,112.54,101.45,68.46,57.81,53.93,53.90,53.75,41.86, 38.88,34.20,33.08,32.32,32.27,30.35,26.27 ,25.83.HRMS(ESI+):calculated for C 35 H 53 Cl 2 N 9 O 4 [M+H] + 734.3670, found 734.3659.
实施例10Example 10
本实施例提供一种氯福克酚衍生物FT60及其制备方法，合成路线如下：This embodiment provides a chlorphenol derivative FT60 and its preparation method. The synthesis route is as follows:
Figure PCTCN2022088513-appb-000033
Figure PCTCN2022088513-appb-000033
中间体FT59：
Figure PCTCN2022088513-appb-000034
Intermediate FT59:
Figure PCTCN2022088513-appb-000034
将氯福克酚(200.0mg，0.55mmol)、碳酸钾(151.3mg，1.09mmol)和溴丁酸乙酯(138μL，1.09mmol)按照合成FT05的方法，制备得到黄色油状产物FT59(223.7mg，88％)。 1H NMR(400MHz,CD 3OD)δ7.38(d,J＝2.0Hz,1H),7.22–7.16(m,1H),7.11–7.05(m,2H),6.92(d,J＝8.3Hz,1H),6.75(d,J＝8.5Hz,1H),4.01(s,2H),3.95(t,J＝5.9Hz,2H),3.67(s,3H),2.36(t,J＝7.3Hz,2H),2.08–1.96(m,2H),1.66(s,2H),1.31(s,6H),0.69(s,9H). 13C NMR(100MHz,CD 3OD)δ173.82,154.43,142.18,137.69,134.83,132.13,131.32,129.11,128.99,126.85,126.29,125.39,110.53,66.65,57.03,51.72,38.01,33.71,32.42,31.89,31.80,30.48,24.74.HRMS(ESI+):calculated for C 26H 34Cl 2O 3[M+H] +465.1958,found 465.1956. The yellow oily product FT59 (223.7 mg, 88%). 1 H NMR (400MHz, CD 3 OD) δ7.38 (d, J＝2.0Hz, 1H), 7.22–7.16 (m, 1H), 7.11–7.05 (m, 2H), 6.92 (d, J＝8.3Hz ,1H),6.75(d,J＝8.5Hz,1H),4.01(s,2H),3.95(t,J＝5.9Hz,2H),3.67(s,3H),2.36(t,J＝7.3Hz ,2H),2.08–1.96(m,2H),1.66(s,2H),1.31(s,6H),0.69(s,9H). 13 C NMR(100MHz,CD 3 OD)δ173.82,154.43,142.18, 137.69,134.83,132.13,131.32,129.11,128.99,126.85,126.29,125.39,110.53,66.65,57.03,51.72,38.01,33.71,32.42,31.89,31.80,30. 48,24.74.HRMS(ESI+):calculated for C 26 H 34 Cl 2 O 3 [M+H] + 465.1958,found 465.1956.
产物FT60：
Figure PCTCN2022088513-appb-000035
Product FT60:
Figure PCTCN2022088513-appb-000035
将反应物FT59(56.3mg，0.12mmol)、HATU(115.3mg，0.30mmol)、DIPEA(105μL，0.60mmol)和H-Arg-Arg-Arg-OMe·4HCl(90.8mg，0.14mmol)按照合成FT07的方法，制备得到黄色油状产物FT60(96.4mg，76％)。 1H NMR(400MHz,CD 3OD)δ7.43(s,1H),7.20(d,J＝8.4Hz,2H),7.11–7.02(m,2H),6.86(d,J＝8.5Hz,1H),4.48–4.27(m,3H),4.03(s,2H),3.98(t,J＝6.1Hz,2H),3.72(s,3H),3.26–3.13(m,6H),2.41(t,J＝7.4Hz,2H),2.09–1.60(m,16H),1.28(s,6H),0.66(s,9H). 13C NMR(100MHz,CD 3OD)δ175.85,174.34,174.07,173.55,170.01,158.71,155.75,142.84,139.15,135.91,133.32,133.08,129.81,129.70,127.99,127.44,126.45,111.82,68.29,57.88,54.56,54.15,53.27,52.86,41.89,41.88,41.78,38.76,34.23,33.23,33.07,32.34,32.27,30.14,29.96,29.45,26.59,26.24,26.17.HRMS(ESI+):calculated for C 44H 70Cl 2N 12O 6[M+H] +933.4991,found933.4985. The reactants FT59 (56.3 mg, 0.12 mmol), HATU (115.3 mg, 0.30 mmol), DIPEA (105 μL, 0.60 mmol) and H-Arg-Arg-Arg-OMe·4HCl (90.8 mg, 0.14 mmol) were synthesized according to the method of synthesizing FT07 Using this method, the yellow oily product FT60 (96.4 mg, 76%) was prepared. 1 H NMR (400MHz, CD 3 OD) δ7.43 (s, 1H), 7.20 (d, J = 8.4Hz, 2H), 7.11–7.02 (m, 2H), 6.86 (d, J = 8.5Hz, 1H ),4.48–4.27(m,3H),4.03(s,2H),3.98(t,J＝6.1Hz,2H),3.72(s,3H),3.26–3.13(m,6H),2.41(t, J＝7.4Hz,2H),2.09–1.60(m,16H),1.28(s,6H),0.66(s,9H). 13 C NMR(100MHz,CD 3 OD)δ175.85,174.34,174.07,173.55,170.01 ,158.71,155.75,142.84,139.15,135.91,133.32,133.08,129.81,129.70,127.99,127.44,126.45,111.82,68.29,57.88,54.56,54.15,53.27 ,52.86,41.89,41.88,41.78,38.76,34.23,33.23 ,33.07,32.34,32.27,30.14,29.96,29.45,26.59,26.24,26.17.HRMS(ESI+):calculated for C 44 H 70 Cl 2 N 12 O 6 [M+H] + 933.4991,found933.4985.
生物实验评估方法Biological Experiment Assessment Methods
1、抗菌活性测定1. Antibacterial activity determination
根据临床和实验室标准协会(CLSI)指南规定的肉汤稀释法测试所合成化合物的抗菌活性。将细菌细胞接种到Mueller-Hinton琼脂(MHA)平板上培养过夜，并用PBS将细菌细胞浓度调整为大约1×10 6CFU/mL，用于制备细菌悬浮液。将样品首先溶解在DMSO/H 2O中，制备浓度为1000μg/mL(DMSO的最终浓度≤2％)的样品储备液，然后用Mueller-Hinton琼脂(MHB)将储备液稀释 至初始浓度200μg/mL，并将样品溶液(100μL)在96孔板中连续稀释，以获得100μg/mL至0.78μg/mL的浓度。接着在96孔板的每个孔中加入细菌悬浮液(100μL)与测试样品溶液(100μL)混合。最后将96孔板在37℃下孵育24小时。测试在0和24h在600nm处的吸光度。将MIC值定义为未见细菌生长所需化合物的最低浓度。所有实验至少进行两次，并实现生物实验的可重复性。 The antibacterial activity of the synthesized compounds was tested according to the broth dilution method specified by Clinical and Laboratory Standards Institute (CLSI) guidelines. Bacterial cells were inoculated onto Mueller-Hinton agar (MHA) plates and cultured overnight, and the bacterial cell concentration was adjusted to approximately 1×10 6 CFU/mL with PBS for preparation of bacterial suspension. The sample was first dissolved in DMSO/H 2 O to prepare a sample stock solution with a concentration of 1000 μg/mL (final concentration of DMSO ≤ 2%), and then the stock solution was diluted with Mueller-Hinton agar (MHB) to an initial concentration of 200 μg/mL. mL, and the sample solution (100 μL) was serially diluted in a 96-well plate to obtain concentrations from 100 μg/mL to 0.78 μg/mL. Then add bacterial suspension (100 μL) and test sample solution (100 μL) to each well of the 96-well plate and mix. Finally, the 96-well plate was incubated at 37°C for 24 hours. Test the absorbance at 600nm at 0 and 24h. The MIC value is defined as the lowest concentration of a compound required for bacterial growth at which no bacterial growth is observed. All experiments were performed at least twice, and reproducibility of biological experiments was achieved.
2、溶血活性测定2. Determination of hemolytic activity
将新鲜的兔红细胞(RBCs)以2500rpm离心3分钟，然后用PBS洗涤两次。随后用PBS重悬兔红细胞，以制备8％(v/v)的细胞悬浮液。将样品首先溶解在DMSO或PBS中(DMSO的终浓度≤0.5％)，然后用PBS稀释以制备两倍梯度稀释液(400至3.125μg/mL)。将兔红细胞悬浮液(100μL)与样品的两倍梯度稀释液(100μL)混合后加入无菌96孔板中，将96孔板在37℃下孵育1小时，并以2500rpm离心5分钟。将上清液(100μL)转移至新的96孔板中，使用多功能酶标仪测定576nm处的吸光度，2％Triton X-100溶液处理组用作阳性对照，PBS或含0.5％DMSO处理组用作阴性对照。通过以下等式计算溶血活性：％溶血活性＝[(Abs 样品–Abs 阴性对照)/(Abs 阳性对照–Abs 阴性对照)]×100。所有实验至少进行两次，并实现生物实验的可重复性。 Fresh rabbit red blood cells (RBCs) were centrifuged at 2500 rpm for 3 min and then washed twice with PBS. Rabbit red blood cells were then resuspended in PBS to prepare an 8% (v/v) cell suspension. Samples were first dissolved in DMSO or PBS (final concentration of DMSO ≤ 0.5%) and then diluted with PBS to prepare two-fold gradient dilutions (400 to 3.125 μg/mL). Rabbit red blood cell suspension (100 μL) was mixed with a two-fold gradient dilution of the sample (100 μL) and added to a sterile 96-well plate. The 96-well plate was incubated at 37°C for 1 hour and centrifuged at 2500 rpm for 5 minutes. Transfer the supernatant (100 μL) to a new 96-well plate, and use a multifunctional microplate reader to measure the absorbance at 576 nm. The 2% Triton X-100 solution treatment group is used as a positive control, and the PBS or 0.5% DMSO treatment group is used. Used as negative control. Hemolytic activity was calculated by the following equation: % hemolytic activity = [(Abs sample – Abs negative control )/(Abs positive control – Abs negative control )] × 100. All experiments were performed at least twice, and reproducibility of biological experiments was achieved.
3、耐药性发展倾向评估3. Assessment of drug resistance development tendency
通过上述MIC测定方法获得化合物FT09、诺氟沙星对金黄色葡萄球菌ATCC29213的初始MIC值以及FT09、阿莫西林对大肠杆菌ATCC25922的初始MIC值。然后取出浓度为0.5×MIC的96孔板中的细菌细胞，以制备细菌悬浮液(约1×10 6CFU/mL)，用于下一步的MIC值测定。将样品在37℃下孵育24小时后，测量样品的MIC值变化。该实验连续进行22-23天。 The initial MIC values of compound FT09 and norfloxacin against Staphylococcus aureus ATCC29213 and the initial MIC values of FT09 and amoxicillin against E. coli ATCC25922 were obtained by the above MIC determination method. Then take out the bacterial cells in the 96-well plate with a concentration of 0.5×MIC to prepare a bacterial suspension (about 1×10 6 CFU/mL) for the next step of MIC value determination. After the sample was incubated at 37°C for 24 hours, the change in MIC value of the sample was measured. The experiment was carried out continuously for 22-23 days.
4、对哺乳动物细胞的毒性测定(CCK-8试验)4. Toxicity determination to mammalian cells (CCK-8 test)
通过CCK-8法评估氯福克酚及其衍生物FT09对小鼠成纤维细胞(NCTC clone 929)的细胞毒性。将对数期的小鼠成纤维细胞(NCTC clone 929)细胞用0.25％胰酶(含EDTA)消化3分钟，然后吹打并用RPMI 1640培养基(含10％胎牛血清FBS)稀释接种至96孔板中(约2×10 4个细胞/孔)，在5％CO 2气氛下于37℃孵育24h后除去培养基。将氯福克酚及其衍生物FT09首先溶解在DMSO(DMSO的终浓度≤0.1％)中，然后用RPMI 1640培养基(含10％胎牛血清FBS)稀释以制备两倍梯度稀释液(200至3.125μg/mL)。将上述样品的两倍梯度稀释液(100μL)分别加入96孔板中与NCTC clone 929细胞共同孵育24h；最后向每孔加入终浓度为10μL的CCK-8溶液，并于培养箱中孵育1.0h后用酶标仪测定溶液在450nm处的吸光度(OD 450)。根据以下公式计算待测样品的细胞毒性：
Figure PCTCN2022088513-appb-000036
A 加样：具有细胞、CCK8溶液、待测样品和培养基的孔的吸光度；A 空白：具有培养基和CCK8溶液而没有细胞的孔的吸光度；A 未加样：具有细胞、CCK8溶液、培养基而没有待测样品的孔的吸光度。实验至少重复两次，并实现生物实验的可重复性。 The cytotoxicity of chlorphenol and its derivative FT09 on mouse fibroblasts (NCTC clone 929) was evaluated by CCK-8 method. Log-phase mouse fibroblasts (NCTC clone 929) cells were digested with 0.25% trypsin (containing EDTA) for 3 minutes, then pipetted and diluted with RPMI 1640 medium (containing 10% fetal bovine serum FBS) and inoculated into 96 wells. plate (approximately 2 × 10 4 cells/well), incubate at 37°C for 24 h under a 5% CO 2 atmosphere and then remove the culture medium. Chlorphenol and its derivative FT09 were first dissolved in DMSO (final concentration of DMSO ≤ 0.1%), and then diluted with RPMI 1640 medium (containing 10% fetal bovine serum FBS) to prepare a two-fold gradient dilution (200 to 3.125μg/mL). Two-fold gradient dilutions (100 μL) of the above samples were added to a 96-well plate and incubated with NCTC clone 929 cells for 24 h; finally, a CCK-8 solution with a final concentration of 10 μL was added to each well and incubated in the incubator for 1.0 h. Then use a microplate reader to measure the absorbance of the solution at 450nm (OD 450 ). Calculate the cytotoxicity of the sample to be tested according to the following formula:
Figure PCTCN2022088513-appb-000036
A Added : The absorbance of the wells with cells, CCK8 solution, sample to be tested and culture medium; A Blank : The absorbance of the wells with culture medium and CCK8 solution but no cells; A Unsampled : With cells, CCK8 solution, culture The absorbance of the well without the sample to be measured. Experiments were repeated at least twice and reproducibility of biological experiments was achieved.
5、SYTOX green实验5. SYTOX green experiment
将细菌细胞在MHB培养基中培育至对数期，然后用PBS缓冲液(10mM，pH＝7.2)洗涤两次，离心得到细菌细胞。然后将细菌细胞重悬于PBS中，并调整细菌悬浮液浓度至吸光度OD 600＝0.2。向细菌悬浮液中加入0.3μM SYTOX Green染料，使用酶标仪(激发波长：504nm，发射波长：523nm)监测其荧光强度变化。待荧光信号稳定后，将不同浓度的样品(1×MIC，2×MIC和4×MIC)加入至上述含SYTOX Green染料的细菌悬浮液中。然后使用酶标仪监测其荧光强度变化约1h。以含0.5％DMSO的PBS溶液作为空白对照组。实验至少进行两次，并实现生物实验的可重复性。 Bacterial cells were cultured in MHB medium to the logarithmic phase, then washed twice with PBS buffer (10 mM, pH=7.2), and centrifuged to obtain bacterial cells. The bacterial cells were then resuspended in PBS, and the concentration of the bacterial suspension was adjusted to the absorbance OD 600 =0.2. Add 0.3 μM SYTOX Green dye to the bacterial suspension, and use a microplate reader (excitation wavelength: 504 nm, emission wavelength: 523 nm) to monitor changes in fluorescence intensity. After the fluorescence signal is stable, samples of different concentrations (1×MIC, 2×MIC and 4×MIC) are added to the above bacterial suspension containing SYTOX Green dye. Then use a microplate reader to monitor changes in fluorescence intensity for about 1 hour. PBS solution containing 0.5% DMSO was used as a blank control group. Experiments were performed at least twice and reproducibility of biological experiments was achieved.
6、体内抗菌功效评估6. Evaluation of antibacterial efficacy in vivo
动物体内抗菌功效评估实验已获得华南农业大学实验动物中心的批准，并 按照中国卫生部的政策进行。本研究使用雌性C57BL6小鼠(6-8周，平均体重20g)。首先建立免疫抑制的小鼠模型用于金黄色葡萄球菌ATCC29213诱导的小鼠角膜感染模型，而铜绿假单胞菌ATCC9027诱导的角膜感染模型则未对小鼠进行免疫抑制。在感染前5天，往小鼠腹膜内注射环磷酰胺(100mg/kg)3次。将接种在Mueller Hinton琼脂(MHA)平板上的细菌细胞(金黄色葡萄球菌ATCC29213)用PBS悬浮，并将细菌悬浮液浓度调节为约5×10 7CFU/mL，以用于角膜感染。通过腹腔注射2.5％的Avertin(500mg/kg)麻醉剂对小鼠进行麻醉，然后用无菌针头对小鼠左眼的角膜进行划痕，并将15μL细菌悬浮液滴到受损的角膜上。感染后一天，将小鼠随机分为三组(每组5只小鼠)，每天局部使用化合物(0.5％FT09、5％葡萄糖溶液，5％万古霉素或0.3％加替沙星)四次，连续给药3天。最后对小鼠进行安乐死，收集受伤的角膜，并通过MHA平板计数法对活菌进行计数。使用SPSS 22.0软件计算P值，并认为P≤0.05为具备统计学显著性。 The in vivo antibacterial efficacy evaluation experiments in animals were approved by the Experimental Animal Center of South China Agricultural University and were conducted in accordance with the policies of the Ministry of Health of China. Female C57BL6 mice (6-8 weeks, average weight 20g) were used in this study. First, an immunosuppressed mouse model was established for the mouse corneal infection model induced by Staphylococcus aureus ATCC29213, while the corneal infection model induced by Pseudomonas aeruginosa ATCC9027 did not require immunosuppression of the mice. Five days before infection, mice were injected intraperitoneally with cyclophosphamide (100 mg/kg) three times. Bacterial cells (Staphylococcus aureus ATCC 29213) seeded on Mueller Hinton agar (MHA) plates were suspended with PBS, and the bacterial suspension concentration was adjusted to approximately 5×10 7 CFU/mL for corneal infection. The mice were anesthetized by intraperitoneal injection of 2.5% Avertin (500 mg/kg) anesthetic, and then the cornea of the left eye of the mouse was scratched with a sterile needle, and 15 μL of bacterial suspension was dropped onto the damaged cornea. One day after infection, mice were randomly divided into three groups (5 mice per group) and compounds (0.5% FT09, 5% glucose solution, 5% vancomycin, or 0.3% gatifloxacin) were topically applied four times daily. , administered continuously for 3 days. Finally, the mice were euthanized, the injured corneas were collected, and viable bacteria were counted by MHA plate counting. SPSS 22.0 software was used to calculate the P value, and P≤0.05 was considered to be statistically significant.
实验结果Experimental results
1、抗菌和溶血活性结果如表1所示。其中化合物FT09对革兰氏阳性菌和阴性菌都表现出非常优异的抗菌活性，MIC值为0.39-3.125μg/mL；同时对兔红细胞展示出非常低的溶血活性，HC 50(裂解50％兔红细胞所需化合物的浓度)值大于200μg/mL。该结果表明化合物FT09具有非常高的膜选择性(HC 50/MIC)。 1. The results of antibacterial and hemolytic activities are shown in Table 1. Among them, compound FT09 shows excellent antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria, with an MIC value of 0.39-3.125 μg/mL; it also exhibits very low hemolytic activity against rabbit red blood cells, with HC 50 (lysing 50% rabbit The concentration of the compound required for red blood cells) value is greater than 200 μg/mL. This result shows that compound FT09 has very high membrane selectivity (HC 50 /MIC).
表1基于氯福克酚衍生物的体外抗菌和溶血活性(μg/mL)Table 1 In vitro antibacterial and hemolytic activities (μg/mL) based on chlorphenol derivatives
Figure PCTCN2022088513-appb-000037
Figure PCTCN2022088513-appb-000037
Figure PCTCN2022088513-appb-000038
Figure PCTCN2022088513-appb-000038
[a]ND＝Not determined.[a]ND＝Not determined.
[b]VAN＝万古霉素[b]VAN＝vancomycin
2、耐药性研究结果：耐药性的发展倾向已成为设计和评估新型抗菌药物的关键考虑因素。如图1所示，经过22-23天连续传代后，化合物FT09的MIC值均未观察到大于4倍的增加。相反，诺氟沙星则迅速地产生耐药性，经过11天连续传代后MIC值升高了256倍；阿莫西林同样产生耐药性，经过15天连续传代后MIC值升高了16倍。这些结果表明，化合物FT09可以有效减缓甚至克服细菌耐药性的产生。2. Resistance research results: The tendency of resistance development has become a key consideration in the design and evaluation of new antibacterial drugs. As shown in Figure 1, after 22-23 days of continuous passage, no greater than 4-fold increase in the MIC value of compound FT09 was observed. On the contrary, norfloxacin developed resistance rapidly, and the MIC value increased by 256 times after 11 days of continuous passage; amoxicillin also developed resistance, and the MIC value increased by 16 times after 15 days of continuous passage. . These results indicate that compound FT09 can effectively slow down or even overcome the development of bacterial resistance.
3、体外细胞毒性测定结果：通过CCK-8试验测定了氯福克酚和FT09对哺乳动物细胞的细胞毒性。如图2所示，氯福克酚对小鼠成纤维细胞(NCTC clone929)的毒性比较明显，其CC 50值为14.9μg/mL。FT09对小鼠NCTC clone 929细胞的CC 50值则高达107.1μg/mL，其安全性比氯福克酚提高7.2倍。在100μg/mL FT09存在情况下，仍观察到有53.2±1.3％的细胞存活，表明FT09对哺乳动物细胞表现出极低的毒性，显示出较高的治疗潜力。 3. In vitro cytotoxicity measurement results: The cytotoxicity of chlorphenol and FT09 to mammalian cells was measured by CCK-8 test. As shown in Figure 2, the toxicity of chlorphenol to mouse fibroblasts (NCTC clone929) is relatively obvious, and its CC 50 value is 14.9 μg/mL. The CC 50 value of FT09 against mouse NCTC clone 929 cells is as high as 107.1 μg/mL, and its safety is 7.2 times higher than that of chlorphenol. In the presence of 100 μg/mL FT09, 53.2±1.3% cell survival was still observed, indicating that FT09 exhibits extremely low toxicity to mammalian cells and shows high therapeutic potential.
4、膜靶向的抗菌机制：利用SYTOX Green试验初步探讨了FT09和细菌细胞膜之间的作用情况。如图3所示，当用FT09处理大肠杆菌ATCC25922时，观察到细菌混合液中的SYTOX Green染料的荧光强度显著地增强，并呈现出浓度依赖的方式。而对照组的细菌经过PBS处理，细菌混合液中的荧光强调则未发生明显的变化。这些结果表明FT09能够通过浓度依赖的方式对细菌细胞膜的完整性造成破坏，诱发细菌细胞内容物的泄露而导致细菌细胞的死亡，初步验证了阳离子型的氯福克酚衍生物FT09具备膜靶向的抗菌机制。4. Membrane-targeted antibacterial mechanism: The SYTOX Green test was used to preliminarily explore the interaction between FT09 and bacterial cell membranes. As shown in Figure 3, when E. coli ATCC25922 was treated with FT09, the fluorescence intensity of the SYTOX Green dye in the bacterial mixture was observed to be significantly enhanced in a concentration-dependent manner. When the bacteria in the control group were treated with PBS, the fluorescence emphasis in the bacterial mixture did not change significantly. These results indicate that FT09 can damage the integrity of bacterial cell membranes in a concentration-dependent manner, inducing the leakage of bacterial cell contents and leading to the death of bacterial cells. This preliminarily verifies that the cationic chlorphenol derivative FT09 has membrane targeting. antibacterial mechanism.
5、体内抗菌活性评估结果：化合物FT09对革兰氏阳性菌和阴性菌均表现出优异的体外抗菌活性，并具备非常高的膜选择性和较高的安全性，我们进一步评估其在动物体内的抗菌功效。在本研究中，首先利用环磷酰胺处理小鼠，对小鼠进行免疫抑制，然后建立金黄色葡萄球菌ATCC29213诱导的小鼠角膜感染模型；其次直接通过铜绿假单胞菌ATCC9027诱导建立小鼠角膜感染模型(未对小鼠进行免疫抑制)。如图4所示，化合物FT09对金黄色葡萄球菌ATCC29213(阳性菌)以及铜绿假单胞菌ATCC9027(阴性菌)都表现出优异的体内抗菌活性，可使受感染角膜中的细菌的减少量都大于99.9％，其疗效可与市售的万古霉素或者加替沙星相互比拟。这些结果表明，化合物FT09能够治愈由金黄色葡萄球菌以及铜绿假单胞菌引起的小鼠角膜感染。5. In vivo antibacterial activity evaluation results: Compound FT09 shows excellent in vitro antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria, and has very high membrane selectivity and high safety. We further evaluate its use in animals. antibacterial effect. In this study, mice were first treated with cyclophosphamide to immunosuppress the mice, and then a mouse corneal infection model induced by Staphylococcus aureus ATCC29213 was established; secondly, a mouse cornea infection model was directly induced by Pseudomonas aeruginosa ATCC9027. Infection model (mice were not immunosuppressed). As shown in Figure 4, compound FT09 showed excellent in vivo antibacterial activity against Staphylococcus aureus ATCC29213 (positive bacteria) and Pseudomonas aeruginosa ATCC9027 (negative bacteria), which can reduce the amount of bacteria in the infected cornea. More than 99.9%, its efficacy is comparable to commercially available vancomycin or gatifloxacin. These results indicate that compound FT09 can cure mouse corneal infections caused by Staphylococcus aureus and Pseudomonas aeruginosa.
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。以上所述实施例仅表达了本发明的几种实施方式，便于具体和详细地理解本发明的技术方案，但并不能因此而理解为对发明专利保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。应当理解，本领域技术人员在本发明提供的技术方案的基础上，通过合乎逻辑的分析、推理或者有限的试验得到的技术方案，均在本发明所附权利要求的保护范围内。因此，本发明专利的保护范围应以所附权利要求的内容为准，说明书及附图可以用于解释权利要求的内容。The technical features of the above-described embodiments can be combined in any way. To simplify the description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, All should be considered to be within the scope of this manual. The above-mentioned embodiments only express several implementation modes of the present invention to facilitate a specific and detailed understanding of the technical solutions of the present invention, but they should not be construed as limiting the scope of protection of the invention patent. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. It should be understood that technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited testing based on the technical solutions provided by the present invention are within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the contents of the appended claims, and the description and drawings may be used to interpret the contents of the claims.

Claims (10)

  1. 一种氯福克酚衍生物，其特征在于，其结构如通式(1)所示：A kind of chlorphenol derivative, characterized in that its structure is shown in the general formula (1):
    Figure PCTCN2022088513-appb-100001
    Figure PCTCN2022088513-appb-100001
    其中：in:
    L 1选自具有1至10个C原子的直链烷基或具有3至10个C原子的支链或环状的烷基； L 1 is selected from a linear alkyl group having 1 to 10 C atoms or a branched or cyclic alkyl group having 3 to 10 C atoms;
    R 1选自胍基、氨基、精氨酸基团、组氨酸基团、赖氨酸基团或这些基团的组合。 R1 is selected from a guanidine group, an amino group, an arginine group, a histidine group, a lysine group or a combination of these groups.
  2. 根据权利要求1所述的氯福克酚衍生物，其特征在于，L 1选自具有1至6个C原子的直链烷基或具有3至6个C原子的支链或环状的烷基。 The chlorphenol derivative according to claim 1, characterized in that, L1 is selected from a linear alkyl group with 1 to 6 C atoms or a branched or cyclic alkyl group with 3 to 6 C atoms. base.
  3. 根据权利要求2所述的氯福克酚衍生物，其特征在于，L 1选自具有1至4个C原子的直链烷基。 The chlorphenol derivative according to claim 2, characterized in that L 1 is selected from a straight-chain alkyl group having 1 to 4 C atoms.
  4. 根据权利要求1所述的氯福克酚衍生物，其特征在于，其结构如通式(2-1)或(2-2)所示：The chlorphenol derivative according to claim 1, characterized in that its structure is represented by the general formula (2-1) or (2-2):
    Figure PCTCN2022088513-appb-100002
    Figure PCTCN2022088513-appb-100002
  5. 根据权利要求1所述的氯福克酚衍生物，其特征在于，R 1选自如下基团的 任一种： The chlorphenol derivative according to claim 1, wherein R1 is selected from any one of the following groups:
    Figure PCTCN2022088513-appb-100003
    Figure PCTCN2022088513-appb-100003
  6. 根据权利要求1所述的氯福克酚衍生物，其特征在于，所述氯福克酚衍生物选自如下结构：The chlorphenol derivative according to claim 1, characterized in that the chlorphenol derivative is selected from the following structures:
    Figure PCTCN2022088513-appb-100004
    Figure PCTCN2022088513-appb-100004
  7. 一种如权利要求1～6任一项所述的氯福克酚衍生物的制备方法，其特征在于，包括如下步骤：A method for preparing the chlorphenol derivative according to any one of claims 1 to 6, characterized in that it includes the following steps:
    将化合物
    Figure PCTCN2022088513-appb-100005
    进行威廉姆逊合成反应，制备中间体A
    Figure PCTCN2022088513-appb-100006
    将所述中间体A进行水解反应，制备中间体B
    Figure PCTCN2022088513-appb-100007
    compound
    Figure PCTCN2022088513-appb-100005
    Carry out Williamson synthesis reaction to prepare intermediate A
    Figure PCTCN2022088513-appb-100006
    The intermediate A is subjected to a hydrolysis reaction to prepare intermediate B
    Figure PCTCN2022088513-appb-100007
    将所述中间体B进行脱水缩合反应，制备所述氯福克酚衍生物。The intermediate B is subjected to a dehydration condensation reaction to prepare the chlorphenol derivative.
  8. 根据权利要求7所述的氯福克酚衍生物的制备方法，其特征在于，威廉姆逊合成反应的温度为60℃～70℃，时间为4h～5h。The preparation method of chlorphenol derivatives according to claim 7, characterized in that the temperature of Williamson synthesis reaction is 60°C to 70°C, and the time is 4h to 5h.
  9. 权利要求1～6任一项所述的氯福克酚衍生物在制备抗菌药物中的应用。Application of the chlorphenol derivative described in any one of claims 1 to 6 in the preparation of antibacterial drugs.
  10. 一种抗菌药物，其特征在于，其组分包括权利要求1～6任一项所述的氯福克酚衍生物、盐以及药学上可接受的辅料。An antibacterial drug, characterized in that its components include the clofol derivative, salt and pharmaceutically acceptable excipients described in any one of claims 1 to 6.
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