CN117695290A - Preparation method and application of copper complex with alkyl chain modification - Google Patents
Preparation method and application of copper complex with alkyl chain modification Download PDFInfo
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- CN117695290A CN117695290A CN202410166462.4A CN202410166462A CN117695290A CN 117695290 A CN117695290 A CN 117695290A CN 202410166462 A CN202410166462 A CN 202410166462A CN 117695290 A CN117695290 A CN 117695290A
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- 150000004699 copper complex Chemical class 0.000 title claims abstract description 37
- 125000000217 alkyl group Chemical group 0.000 title claims abstract description 29
- 230000004048 modification Effects 0.000 title claims abstract description 26
- 238000012986 modification Methods 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 23
- 239000003814 drug Substances 0.000 claims abstract description 10
- 239000003053 toxin Substances 0.000 claims abstract description 9
- 231100000765 toxin Toxicity 0.000 claims abstract description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 57
- 150000001875 compounds Chemical class 0.000 claims description 28
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 18
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 14
- ODWXUNBKCRECNW-UHFFFAOYSA-M bromocopper(1+) Chemical compound Br[Cu+] ODWXUNBKCRECNW-UHFFFAOYSA-M 0.000 claims description 13
- 238000010992 reflux Methods 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 10
- 229910052786 argon Inorganic materials 0.000 claims description 9
- 239000012043 crude product Substances 0.000 claims description 9
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 238000001704 evaporation Methods 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 239000000022 bacteriostatic agent Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 20
- 230000000694 effects Effects 0.000 abstract description 12
- 241000894006 Bacteria Species 0.000 abstract description 10
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 6
- 206010059866 Drug resistance Diseases 0.000 abstract description 5
- 230000002209 hydrophobic effect Effects 0.000 abstract description 5
- 229910052751 metal Inorganic materials 0.000 abstract description 5
- 239000002184 metal Substances 0.000 abstract description 5
- 210000000170 cell membrane Anatomy 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 4
- 230000004071 biological effect Effects 0.000 abstract description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 230000012010 growth Effects 0.000 abstract description 3
- 150000003904 phospholipids Chemical class 0.000 abstract description 3
- 239000005749 Copper compound Substances 0.000 abstract description 2
- 150000001880 copper compounds Chemical class 0.000 abstract description 2
- 230000008384 membrane barrier Effects 0.000 abstract description 2
- 230000000149 penetrating effect Effects 0.000 abstract description 2
- 239000010949 copper Substances 0.000 description 46
- 239000000543 intermediate Substances 0.000 description 27
- 230000005764 inhibitory process Effects 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 10
- 229910052799 carbon Inorganic materials 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 8
- 108700012359 toxins Proteins 0.000 description 7
- 238000003756 stirring Methods 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 101710092462 Alpha-hemolysin Proteins 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 229910052802 copper Inorganic materials 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 3
- 238000000921 elemental analysis Methods 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 239000012065 filter cake Substances 0.000 description 3
- 238000004896 high resolution mass spectrometry Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 150000004696 coordination complex Chemical class 0.000 description 2
- 150000001879 copper Chemical class 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- MYMSJFSOOQERIO-UHFFFAOYSA-N 1-bromodecane Chemical compound CCCCCCCCCCBr MYMSJFSOOQERIO-UHFFFAOYSA-N 0.000 description 1
- MNDIARAMWBIKFW-UHFFFAOYSA-N 1-bromohexane Chemical compound CCCCCCBr MNDIARAMWBIKFW-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 208000003100 Pseudomembranous Enterocolitis Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000032770 biofilm formation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- -1 copper metal complex Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229960003085 meticillin Drugs 0.000 description 1
- 230000003448 neutrophilic effect Effects 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 208000008494 pericarditis Diseases 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Classifications
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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
- A61K31/33—Heterocyclic compounds
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
-
- 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
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F1/00—Compounds containing elements of Groups 1 or 11 of the Periodic Table
- C07F1/005—Compounds containing elements of Groups 1 or 11 of the Periodic Table without C-Metal linkages
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Communicable Diseases (AREA)
- Epidemiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of antibacterial medicines, and particularly relates to a preparation method and application of a copper complex with alkyl chain modification. The copper complex has a structure shown in a formula I. Compared with the traditional small organic molecules, the hydrophobic side chain of the copper complex provided by the invention can act with a bacterial cell membrane phospholipid bilayer, so that the transmembrane effect and the detention effect are enhanced, and the biological effect of the metal copper compound can completely enter the interior of bacteria by penetrating through a bacterial biological membrane barrier. According to the experiment of the embodiment of the invention, the copper complex with alkyl chain modification can effectively inhibit the growth of staphylococcus aureus and the formation of a biological film and the generation of alpha-hemolytic toxin thereof at the content of 0.39-1.56 mug/mL, and the copper complex does not trigger the drug resistance tendency of bacteria. Therefore, the alkyl chain modified copper complex provided by the invention has a certain potential in bacteriostasis.
Description
Technical Field
The invention belongs to the technical field of antibacterial medicines, and particularly relates to a preparation method and application of a copper complex with alkyl chain modification.
Background
Staphylococcus aureus [ (S.aureus ]S.aureus) Is the most common pathogenic bacteria in clinical infectious diseases, and can cause the infection of a plurality of parts such as skin soft tissues, blood systems, lower respiratory tracts and the like, thereby causing a series of diseases such as pericarditis, pseudomembranous enteritis, pneumonia, septicemia and the like. Due to the emergence of resistant strains such as methicillin-resistant staphylococcus aureus (MRSA), they are severely resistant to many antibiotics, resulting in challenges in the treatment of such infections.
Furthermore, the development of biofilms in chronic infections is an important causative factor, which involves a variety of microorganisms, and thus, infection associated with biofilms also presents serious challenges. Generally, bacteria in a biofilm are encapsulated in a hydrated matrix of Extracellular Polymeric Substances (EPS) consisting of polysaccharides, proteins, lipids and nucleic acids, which have mechanical stability and together provide immobilization of biofilm-producing bacterial cells, enhancing adhesion of the bacteria to the surface, thus forming stable three-dimensional (3D) connective tissue protecting microorganisms from external influences. Under normal conditions, biofilms are resistant to attack by most chemical bactericides, mainly phagocytic and neutrophilic cells, and survive therein, and it is this drug resistance mechanism of bacteria that results in reduced susceptibility of bacteria to antibiotics, so how to eliminate the biofilm barrier is one of the key points for antibiotic development.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a preparation method and application of a copper complex with alkyl chain modification, and the preparation method specifically adopts the following technical scheme:
in a first aspect of the present invention, there is provided a copper complex having an alkyl chain modification, which can be used in the preparation of a medicament for inhibiting staphylococcus aureus, the copper complex having a structure represented by formula I:
a formula I; wherein n is selected from any positive integer of 3,7 and 11.
Preferably, the copper complex has a structure as shown in formula II:
formula II.
Simple substance and oxide forms of copper have been widely used as antibacterial agents, the invention obtains a metallic copper complex through multifunctional organic combination, the complex can effectively resist bacterial drug resistance and inhibit bacterial biofilm, and experiments prove that the metallic copper has good advantages in terms of antibacterial performance and lower cost, and can effectively inhibit the formation of bacterial biofilm. According to the invention, the hydrophobic alkyl side chain can be effectively inserted into a phospholipid bilayer of a bacterial cell membrane, and the metal copper complex enables substances in the cell to leak, so that the cell dies. And the multi-coordination configuration of the metal complex enables the metal complex to be modified by different ligands, so that the better biological activity effect is achieved. Therefore, the damage to bacterial biomembrane, or even elimination, can be achieved by modifying the metallic copper complex, so that the drug-resistant strain is killed.
The second aspect of the invention also provides a preparation method of the copper complex with alkyl chain modification, which comprises the following steps:
s1: under the argon condition, heating and refluxing the compound with the structure of formula I-a, the compound with the structure of formula I-d and potassium carbonate in acetonitrile, cooling to room temperature, decompressing and evaporating the solvent to obtain a crude product, and purifying to obtain an intermediate with the structure of formula I-e;
s2: under the argon condition, heating and refluxing the compound with the structure of formula I-b, the compound with the structure of formula I-d and potassium carbonate in acetonitrile, cooling to room temperature, decompressing and evaporating the solvent to obtain a crude product, and purifying to obtain an intermediate with the structure of formula I-f;
s3: under the argon condition, heating and refluxing the compound with the structure of formula I-c, the compound with the structure of formula I-d and potassium carbonate in acetonitrile, cooling to room temperature, decompressing and evaporating the solvent to obtain a crude product, and purifying to obtain an intermediate with the structure of formula I-g;
s4: dissolving an intermediate with a structure of formula I-e or an intermediate with a structure of formula I-f or an intermediate with a structure of formula I-g in acetonitrile solution, then adding the acetonitrile solution of copper bromide, reacting for 6 hours at normal temperature, filtering, washing and drying to obtain a copper complex with alkyl chain modification;
wherein the molecular structural formulas of the compound with the structure of formula I-a, the compound with the structure of formula I-b, the compound with the structure of formula I-c, the compound with the structure of formula I-d, the intermediate with the structure of formula I-e, the intermediate with the structure of formula I-f and the intermediate with the structure of formula I-g are shown as follows:
。
preferably, the molar ratio of the compound of formula I-a, the compound of formula I-b and the compound of formula I-c to the compound of formula I-d in steps S1, S2 and S3 is 1:1, a step of; purification in steps S1, S2 and S3 above was performed on an alumina column with dichloromethane/methanol=200: 1, eluting and purifying by using an eluent; the temperature of the heating reflux in the steps S1, S2 and S3 is 85 ℃; the molar ratio of the intermediate of the structure of the formula I-e, the intermediate of the structure of the formula I-f and the intermediate of the structure of the formula I-g in the step S4 to copper bromide is 1:2.
the invention also provides an application of the copper complex with alkyl chain modification in preparing a medicine for inhibiting alpha-hemolysin.
The invention also provides an application of the copper complex with alkyl chain modification in preparing a bacteriostatic agent.
The beneficial effects of the invention are as follows: the invention provides a copper complex with alkyl chain modification, wherein a hydrophobic side chain of the copper complex can act with a bacterial cell membrane phospholipid bilayer, compared with a traditional small organic molecule, the copper complex has the advantages that the transmembrane effect and the detention effect are enhanced, and the biological effect of a metal copper compound can completely enter the inside of bacteria through penetrating a bacterial biological membrane barrier. According to the experiment of the embodiment of the invention, the copper complex with alkyl chain modification can effectively inhibit the growth of staphylococcus aureus and the formation of a biological film and the generation of alpha-hemolytic toxin thereof at the content of 0.39-1.56 mug/mL, and the copper complex does not trigger the drug resistance tendency of bacteria. Therefore, the alkyl chain modified copper complex provided by the invention has a certain potential in bacteriostasis.
Drawings
FIG. 1 shows intermediates of formula I-e, formula I-f, formula I-g and complex C 6 -Cu、C 10 -Cu、C 14 -MIC profile of Cu against staphylococcus aureus;
FIG. 2 shows complex C 10 -a Cu inhibition biofilm assay for staphylococcus aureus;
FIG. 3 shows complex C 10 -a graph of Cu inhibition of s.aureus by α -hemolysin;
FIG. 4 shows Staphylococcus aureus vs. Complex C 10 -Cu resistance profile;
Detailed Description
The conception, specific structure, and technical effects produced by the present invention will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects, and effects of the present invention. It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other.
Example 1
A copper complex with alkyl chain modification, the preparation method comprising the steps of:
the reaction is shown in the following reaction scheme:
(1) Complex C 6 Preparation of Cu
Step 1: the preparation of the intermediate of the structure of the formula I-e, wherein the structural formula of the intermediate I-e is as follows:
the specific process is as follows: 1-Bromohexane (0.5 mmoL, 82.5 mg), 2' -dipyridylmethylamine (0.5 mmoL,100 mg), potassium carbonate (1.5 mmoL, 207 mg) and acetonitrile (2 mL) in a 25mL round bottom reaction flask were heated to reflux at 85℃under argon for 24 hours, cooled to room temperature after completion of the reaction, filtered to remove excess carbonate, and the crude product was purified by column chromatography on neutral alumina with dichloromethane-methanol mixture (200:1, v/v) as eluent, solvent removed under reduced pressure and dried in vacuo to give intermediate I-e as a yellow oil, yield: 94mg,66.33%.
Step 2: complex C 6 Preparation of Cu, complex C 6 -Cu has the structural formula:
the specific process is as follows: weighing copper bromide (0.2 mmoL, 45 mg) in a 25mL round bottom reaction bottle, adding 2mL acetonitrile, stirring thoroughly to obtain copper bromide solution, slowly dripping acetonitrile solution of intermediate I-e (0.1 mmoL, 28 mg) into copper bromide solution under stirring, reacting at normal temperature for 6h, filtering, collecting filter cake, washing with water for three times, vacuum drying to obtain complex C 6 -Cu as green solid, yield: 28mg,55.6%.
The characterization result is as follows:
high resolution mass spectrometry (ESI) m/z: c (C) 18 H 25 BrCuN 3 Theoretical value 425.0528, [ M-Br ]] + Is 425.0519. Elemental analysis (%): c (C) 18 H 25 Br 2 CuN 3 (506.8) theory C42.66, H4.97, N8.29, found C42.54, H4.74, N8.26.
(2) Complex C 10 Preparation of Cu
Step 1: the preparation of the intermediate of the structure of the formula I-f, wherein the structural formula of the intermediate I-f is as follows:
the specific process is as follows: 1-Bromodecane (0.5 mmoL, 118 mg), 2' -dipyridylmethylamine (0.5 mmoL,100 mg), potassium carbonate (1.5 mmoL, 207 mg) and acetonitrile (2 mL) in a 25mL round bottom flask were heated to reflux at 85℃under argon for 24 hours, cooled to room temperature after completion of the reaction, filtered to remove excess carbonate, and the crude product was purified by column chromatography on neutral alumina using a dichloromethane-methanol mixture (200:1, v/v) as eluent, the solvent was removed under reduced pressure and dried in vacuo to give intermediate I-f as a yellow oil, yield: 145.5mg,85.57%.
Step 2: complex C 10 Preparation of Cu, complex C 10 -Cu has the structural formula:
the specific process is as follows: weighing copper bromide (0.2 mmoL, 45 mg) in a 25mL round bottom reaction bottle, adding 2mL acetonitrile, stirring thoroughly to obtain copper bromide solution, slowly dripping acetonitrile solution of intermediate I-f (0.1 mmoL, 34 mg) into copper bromide solution under stirring, reacting at normal temperature for 6h, filtering, collecting filter cake, washing with water for three times, vacuum drying to obtain complex C 10 -Cu as green solid, yield: 33mg,58.9%.
The characterization result is as follows:
high resolution mass spectrometry (ESI) m/z: c (C) 22 H 33 BrCuN 3 Theoretical value 481.1154, [ M-Br ]] + Is 481.1148. Elemental analysis (%): c (C) 22 H 33 Br 2 CuN 3 (562.0) theory C46.94, H5.91, N7.47, found C47.44, H5.88, N7.46.
(3) Complex C 14 Preparation of Cu
Step 1: the preparation of intermediates of the structure of formula I-g, wherein the structural formula of the intermediates I-g is as follows:
the specific process is as follows: 1-Broomtetradecane (0.5 mmoL, 140 mg), 2' -dipyridylmethylamine (0.5 mmoL,100 mg), potassium carbonate (1.5 mmoL, 207 mg) and acetonitrile (2 mL) in a 25mL round bottom reaction flask were heated to reflux at 85℃under argon for 24 hours, cooled to room temperature after completion of the reaction, filtered to remove excess carbonate, and the crude product was purified by column chromatography on neutral alumina using a dichloromethane-methanol mixture (200:1, v/v) as eluent, the solvent was removed under reduced pressure and dried in vacuo to give intermediate I-g as a yellow oil, yield: 166.2mg,84.02%.
Step 2: complex C 14 Preparation of Cu, complex C 14 -Cu has the structural formula:
the specific process is as follows: weighing copper bromide (0.2 mmoL, 45 mg) in a 25mL round bottom reaction bottle, adding 2mL acetonitrile, stirring thoroughly to obtain copper bromide solution, slowly dripping acetonitrile solution of intermediate I-g (0.1 mmoL, 40 mg) into copper bromide solution under stirring, reacting at normal temperature for 6h, filtering, collecting filter cake, washing with water for three times, vacuum drying to obtain complex C 14 -Cu as green solid, yield: 34.1mg,55.4%.
The characterization result is as follows:
high resolution mass spectrometry (ESI) m/z: c (C) 26 H 41 BrCuN 3 Theoretical value 537.1780, [ M-Br ]] + Is 537.1778. Elemental analysis (%): c (C) 26 H 41 Br 2 CuN 3 (622.9) 1% C 26 H 41 N 3 ) Theoretical values C50.63, H6.70, N6.81, measured values C51.12, H6.66, N6.71.
Example 2
This example shows the complex C prepared in example 1 6 Cu, complex C 10 Cu and Complex C 14 -Cu in vitro antibacterial Activity test
(1) MIC determination by micropipette assay: the staphylococcus aureus Newman strain was cultured in TSB medium until OD 600 Reaching 1; bacterial cell numbers were diluted to approximately 5X 10 with fresh TSB medium 6 CFU/mL（OD 600 =0.05); subsequently, 50. Mu.L of a copper metal complex or intermediate of the formula I-e, formula I-f, formula I-g are added to200. In bacterial suspension of μl, the final drug concentration at each well is then, in order from left to right: 200. mu g/mL,100 mu g/mL,50 mu g/mL,25 mu g/mL,12.5 mu g/mL,6.25 mu g/mL,3.125 mu g/mL,1.56 mu g/mL,0.78 mu g/mL,0.39 mu g/mL,0.195 mu g/mL, wherein 50 mu L of sterile water is added to the last hole as a blank; after placing the mixture in a 96-well plate and further incubating at 37 ℃ for 20 hours, the results were observed; the turbidity of the well plate was observed, wherein the lowest drug concentration corresponding to the clarified drug administration well was MIC (minimum inhibitory concentration), and the results are shown in fig. 1. Rows a and H are at the edges and no reagent was added to prevent contamination for testing.
FIG. 1 shows intermediates of formula I-e, formula I-f, formula I-g and complex C 6 -Cu、C 10 -Cu、C 14 -MIC profile of Cu against staphylococcus aureus; judging the complex C by observing the turbidity degree of the pore plate 6 -Cu、C 10 -Cu、C 14 -Cu has an antibacterial effect against staphylococcus aureus, wherein the minimum drug concentration corresponding to the clarified dosing well is MIC (minimum inhibitory concentration); and as can be seen from FIG. 1, C 6 -Cu、C 10 -Cu、C 14 Cu has a certain antibacterial effect, wherein the complex C with the shortest alkyl chain 6 Cu has poor antibacterial activity, increases antibacterial activity along with the increase of the hydrophobic alkyl side chain, but when the length of the hydrophobic carbon chain exceeds 10, the activity is reduced to a certain extent, and C 10 Cu shows the best antimicrobial activity and its mic=1.56 μg/mL against staphylococcus aureus.
(2) Inhibition of biofilm effects was determined by biofilm experiments: staphylococcus aureusNewmanStrains were grown in TSB medium for 5 hours, then the cultures were diluted 1:200 with fresh sterile TSB medium containing 0.5% and 24-well microtiter plates were filled with aliquots of bacterial cultures with or without metal copper complexes of 2 mL; plates were incubated at 37℃for 48 hours, then washed 3 times with PBS, 24-well plates were dried overnight at room temperature, the biofilm attached to the microtiter plates was incubated with 0.1% crystal violet solution for 15 minutes, excess crystal violet was removed by washing with PBS, and crystal violet attached to the biofilm samples was usedAcetic acid was dissolved and absorbance at 595, 595 nm was measured to indicate biofilm formation, the results of which are shown in figure 2.
FIG. 2 shows complex C 10 -a Cu inhibition biofilm assay for staphylococcus aureus; by measuring OD 595 Absorbance at the time of determination of C 10 -amount of biofilm after Cu acts on bacteria; and as can be seen from FIG. 2, C 10 The Cu has strong inhibition effect on the biomembrane of staphylococcus aureus, and the absorbance value is reduced by 39 percent when being compared with a control group at the time of 0.39 mu g/mL, and the inhibition effect of 41.8 percent and 56.8 percent on the growth of cell membranes are respectively shown by 0.78 mu g/mL and 1.56 mu g/mL, which indicates that C 10 Cu can inhibit the production of bacterial biofilm to some extent.
(3) Inhibition of alpha-hemolytic toxin was determined by rabbit erythrocyte hemolysis rate: staphylococcus aureusNewmanThe strain was cultured in TSB medium for 5 hours. The bacteria were then diluted 1000-fold in fresh TSB medium. A 24-well microtiter plate was filled with 2mL aliquots of bacterial cultures with or without metal copper complexes. Plates were incubated at 37℃for 10 hours. Subsequently, it was centrifuged, and the bacterial supernatant was retained. 1.1 mL sterile PBS, 50 μl rabbit red blood cells, 50 μl bacterial supernatant were added to a 1.5mLep tube, followed by incubation at 37deg.C for 30 minutes. After incubation was completed, centrifugation (2000 rmp,2 min) and the supernatant was assayed at OD 540 The absorbance at nm is shown in FIG. 3.
FIG. 3 shows complex C 10 -a graph of Cu inhibition of s.aureus by α -hemolysin; by measuring OD 540 Determination of C by absorbance at nm 10 The inhibition of bacterial alpha-hemolytic toxin production after Cu treatment, the higher the absorbance value, the more the rabbit red blood cells are ruptured, i.e. the more the bacterial alpha-hemolytic toxin is produced, leading to toxin hemolysis; and as can be seen from FIG. 3, C 10 The Cu has a strong inhibition effect on the generation of staphylococcus aureus alpha-hemolytic toxin, and the absorbance value is reduced by 42.1% and 45.7% when the absorbance value is 0.39 mug/mL and 0.78 mug/mL compared with a control group. Whereas 1.56 μg/mL showed 86.3% inhibition of alpha-hemolysin production, indicating C 10 Cu can inhibit alpha-dissolution to a certain extentProduction of blood toxins.
(4) Drug resistance study: first, in the case of the para-complex C 10 -MIC determination of Cu against staphylococcus aureus, wherein the MIC value is at day 0, the MIC value determination method is determined by the aforementioned microdilution method; then, 5. Mu.L of complex C at a 1/2 XMIC concentration was transferred 10 Re-incubation of Cu bacterial cultures in 5mL fresh broth medium to log phase, re-determination of MIC values, repeating the procedure 20 times and recording each MIC value, the MIC value of each day divided by the MIC value of day 0 yields fold-change values.
FIG. 4 is a graph showing the determination of the resistance of Staphylococcus aureus to complex C10-Cu; whether resistance developed was observed by observing changes in MIC values of staphylococcus aureus. If resistance develops, the MIC of Staphylococcus aureus becomes large, indicating that the concentration at the first administration is insufficient to inhibit Staphylococcus aureus growth. If no resistance is developed, the MIC value will not become large. As can be seen from fig. 4, after 20 determinations, the MIC value of staphylococcus aureus did not increase, indicating that staphylococcus aureus was not resistant.
While the present invention has been described in considerable detail and with particularity with respect to several described embodiments, it is not intended to be limited to any such detail or embodiments or any particular embodiment, but is to be construed as providing broad interpretation of such claims by reference to the appended claims in view of the prior art so as to effectively encompass the intended scope of the invention. Furthermore, the foregoing description of the invention has been presented in its embodiments contemplated by the inventors for the purpose of providing a useful description, and for the purposes of providing a non-essential modification of the invention that may not be presently contemplated, may represent an equivalent modification of the invention.
Claims (9)
1. The application of a copper complex with alkyl chain modification in preparing a medicament for inhibiting staphylococcus aureus is characterized in that the copper complex with alkyl chain modification has a structure shown in a formula I:
a formula I; wherein n is selected from any positive integer of 3,7 and 11.
2. The use according to claim 1, wherein the copper complex with alkyl chain modification has the structure shown in formula II:
formula II.
3. A process for the preparation of a copper complex having an alkyl chain modification, characterized in that the copper complex having an alkyl chain modification has the structure of formula I in claim 1, comprising the steps of:
s1: under the argon condition, heating and refluxing the compound with the structure of formula I-a, the compound with the structure of formula I-d and potassium carbonate in acetonitrile, cooling to room temperature, decompressing and evaporating the solvent to obtain a crude product, and purifying to obtain an intermediate with the structure of formula I-e;
s2: under the argon condition, heating and refluxing the compound with the structure of formula I-b, the compound with the structure of formula I-d and potassium carbonate in acetonitrile, cooling to room temperature, decompressing and evaporating the solvent to obtain a crude product, and purifying to obtain an intermediate with the structure of formula I-f;
s3: under the argon condition, heating and refluxing the compound with the structure of formula I-c, the compound with the structure of formula I-d and potassium carbonate in acetonitrile, cooling to room temperature, decompressing and evaporating the solvent to obtain a crude product, and purifying to obtain an intermediate with the structure of formula I-g;
s4: dissolving an intermediate with a structure of formula I-e or an intermediate with a structure of formula I-f or an intermediate with a structure of formula I-g in acetonitrile solution, then adding the acetonitrile solution of copper bromide, reacting for 6 hours at normal temperature, filtering, washing and drying to obtain a copper complex with alkyl chain modification;
wherein the molecular structural formulas of the compound with the structure of formula I-a, the compound with the structure of formula I-b, the compound with the structure of formula I-c, the compound with the structure of formula I-d, the intermediate with the structure of formula I-e, the intermediate with the structure of formula I-f and the intermediate with the structure of formula I-g are shown as follows:
。
4. the process according to claim 3, wherein the molar ratio of the compound of formula I-a, the compound of formula I-b and the compound of formula I-c to the compound of formula I-d in steps S1, S2 and S3 is 1:1.
5. a process according to claim 3, wherein the purification in steps S1, S2 and S3 is performed on an alumina column with dichloromethane/methanol = 200:1, eluting and purifying by using an eluent.
6. A process according to claim 3, wherein the temperature of the heated reflux in steps S1, S2 and S3 is 85 ℃.
7. The method according to claim 3, wherein the molar ratio of the intermediate of the structure of formula I-e, the intermediate of the structure of formula I-f and the intermediate of the structure of formula I-g to copper bromide in step S4 is 1:2.
8. use of a copper complex with alkyl chain modification in the preparation of a medicament for inhibiting alpha-hemolytic toxin, wherein the copper complex with alkyl chain modification has a structure shown in formula I in claim 1.
9. Use of a copper complex with alkyl chain modification in the preparation of a bacteriostatic agent, characterized in that the copper complex with alkyl chain modification has a structure shown in formula I in claim 1.
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