CN106377527B - Open chain pyridine carboxylic acid derivatives H2Application of the dedpa in antibacterial field - Google Patents
Open chain pyridine carboxylic acid derivatives H2Application of the dedpa in antibacterial field Download PDFInfo
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- CN106377527B CN106377527B CN201610801773.9A CN201610801773A CN106377527B CN 106377527 B CN106377527 B CN 106377527B CN 201610801773 A CN201610801773 A CN 201610801773A CN 106377527 B CN106377527 B CN 106377527B
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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
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
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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
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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Abstract
The invention belongs to field of medicinal chemistry, disclose open chain pyridine carboxylic acid derivatives H2Application of the dedpa in antibacterial field.The compound has the following structure, and can restore to produce metallo-β-lactamase enterobacteriaceae lactobacteriaceae to the sensibility of carbapenem antibiotic.Antimicrobial test is the results show that the escherichia coli (producing NDM-4 type metallo-β-lactamase) of Carbapenem-resistant can be reduced about 40960 times to the MIC value of Meropenem by its highest.Erythrocyte in vitro toxicity test also demonstrates the RBC Toxicity that this compound has very little, and therefore, which can be used as the drug candidate of novel metal beta-lactamase inhibitor.
Description
Technical field
The invention belongs to field of pharmaceutical chemistry technology, it is related to the open chain pyridine carboxylic acid derivatives with potential using value
H2Application of the dedpa as novel metal beta-lactamase inhibitor in antibacterial field.
Background technique
The appearance of antibiotic is that clinical treatment infectious diseases brings Gospel, but against more and more antibiotic
After class drug is developed and applies to clinic, antibiotic brings weakness for the treatment of clinical infectious disease, clinically opens
Begin various endurance strains occur, more and more antibiotic lose initial effect, global Drug resistance status for treatment
Do not allow to despise.2009, the Walsh seminar of Cardiff University, Britain reported a kind of novel beta-lactamase-metal
Beta-lactamase, case show the patient in December, 2007, after India New Delhi receives anti-body extract for treating, the training of urine
Feeding object isolates a kind of Klebsiella Pneumoniae for Nosocomial infection, so this enzyme is known as New Delhi beta-lactam again
Enzyme (Antimicrobial agents and chemotherapy 2009,53,5046.).Have after being reported 1 year from it
Nearly 20 countries report that discovery carries bla in successionNDM-1, serious (the The Lancet Infectious of worldwide trend
Diseases 2010,10, 597.)。
According to the difference of amino acid sequence homology in beta-lactam enzyme molecular structure, Ambler classification is by beta-lactam
Enzyme is divided into tetra- class of A, B, C and D, wherein being classified as serine class beta-lactamase and gold again since its terminal amino group residual is different
Belong to beta-lactamase.Wherein A, C and D belong to serine class beta-lactamase, and B class belongs to metal beta-lactamase.At present on
The beta-lactamase inhibitor in city has clavulanic acid, Sulbactam and Tazobactam Sodium.It can be mentioned with cephalosporin antibacterial combination
The activity of high antibacterials.But it belongs to serine class beta-lactamase inhibitor, and related metallo-β-lactamase presses down
Preparation is reported currently without marketed drug.
2014, Gerard D.Wright et al. reported the natural compound AMA of one kind obtained in the fungi, it
In the case where the activity containing NDM-1 type metallo-β-lactamase, with Meropenem drug combination can quickly and effectively being inhibited
NDM-1 enzyme bacterial strain can be made to produce to Meropenem recovery sensitivity (Nature 2014,510,503.).2015, Sabiha
Y.Essack et al. reports two kinds of Zinc Ions Chelateds agent NOTA and DOTA, this two kinds of chelating agents can carry metal β-interior acyl
The bacterial strain of amine enzyme restores sensibility (the Journal of Antimicrobial to carbapenem antibiotic
Chemotherapy 2015,70,1594.).2015, Yang Kewu seminar, Northwest University reported a series of thioacetic acid sulphur
Ester amino acid derivativges, for the IC of metallo-β-lactamase L150Minimum can achieve 18nM, but its external activity is tested
Prove its restore Meropenem sensitivity effect it is unsatisfactory (Acs Medicinal Chemistry Letters 2015,6,
660.)。
H2Dedpa is the open chain pyridine carboxylic that Teresa Rodr í guez-Blas seminar, Spain in 2008 invents for the first time
Acids ligand is used for divalent zinc ion, coordination (the Dalton transactions of bivalent chromium ion and lead (II) ion
2008,5754.).This compound is used for Chris Orvig seminar, the U.S. in 2010 and radioactive element Ga68 is coordinated,
For positron emission tomography (PET) preparation (Journal of the American Chemical Society
2010,132,15726.).It has the following structure:
Summary of the invention
The purpose of the present invention is to provide H2Dedpa answering in antibacterial field as novel metal beta-lactamase inhibitor
With.
Purpose to realize the present invention, technical solution are as follows: the present invention is to compound H2Dedpa erythrocyte in vitro hemolytic into
Experiment is gone；To compound H2The external Hela of dedpa (drawing cancer cell line in sea) cytotoxicity is tested；To compound
H2Dedpa in vitro tests the sterilization dynamics of escherichia coli 14-55；And H is carried out2Dedpa and NOTA are in body
Outside with Meropenem (MEM), Zn2+Combination comparison.As a result, it has been found that H2Dedpa, can as acyclic chelating, nitrogen heterocycles derivative
Restore so as to produce metallo-β-lactamase bacterial strain to carbapenem antibiotic sensibility.It can be used as novel metal beta-lactam
Enzyme inhibitor is applied in antibacterial field.
Advantage of the present invention and innovative point are: having found compound H2The new application of dedpa, will be used for compound ligand and
The H of positron emission tomography (PET) preparation2Dedpa is as novel metal beta-lactamase inhibitor in antibacterial field
It is applied, and it was found that compound H2Dedpa and Meropenem combination have good bactericidal effect, and find compound
H2Zinc ion in dedpa and metallo-β-lactamase, which combines, causes the enzyme to inactivate, and has invertibity.The application is beneficial to out
Send out novel metal beta-lactamase inhibitor.
Detailed description of the invention
Fig. 1 is compound H2The experimental result of dedpa erythrocyte in vitro hemolytic, as seen from the figure, compound H2Dedpa exists
Erythrocyte hemolysis rate is not damaged red blood cell significantly, less than 1% it is possible thereby to prove it for lactation when 1000 μ g/mL
The Erythrocyte Damage very little of animal.
Fig. 2 is compound H2Dedpa is in vitro for the experimental result of Hela cytotoxicity, as seen from the figure, compound
H2Dedpa in 1000 μ g/mL to the inhibiting rate of Hela cell 48.71%, it is possible thereby to which preliminary proof, was tested in vitro
Cheng Zhong, compound H2Dedpa has lesser cytotoxicity.
Fig. 3 is compound H2Dedpa in vitro for the work dead cell fluorescence experiments of Hela cytotoxicity as a result, in figure,
A-c negative control (not dosing group), d-f compound H2Dedpa (128 μ g/mL), g-i compound H2Dedpa (64 μ g/mL), j-
L positive controls 0.1%Triton X-100.Figure medium scale is 100nm.It can significantly be observed by figure, compound
H2Dedpa maintains the growth of normal morphology to have little effect after 128 μ g/mL act on Hela cell, for cell.
Fig. 4 is compound H2Dedpa is in vitro for the sterilization kinetic results comparison diagram of escherichia coli 14-55, figure
In, a is compound H2Dedpa, b are control compound NOTA, and c is results of turbidity, and in c figure, 1 is blank control, and 2 be chemical combination
Object H2Dedpa (0.125 μ g/mL), 3 be compound H2dedpa(0.25μg/mL).By figure a, b it is found that working as compound H2dedpa
After two concentration effects for 24 hours of 4 × MIC and 8 × MIC, there is good bactericidal activity for the bacterium at logarithmic growth initial stage.With sun
Property control NOTA activity it is relatively excellent.From c figure, for 24 hours after appearance it will be clear that compound H2Dedpa effect
Bacterium solution turbidity afterwards is very low, and blank control group turbidity is obviously big.
Fig. 5 is compound H2Dedpa and NOTA in vitro with Meropenem (MEM), Zn2+Result histogram after combination,
As seen from the figure, compound H2It almost can achieve 100% inhibiting rate after dedpa and Meropenem combination 16-18h, still
Rear drug failure is used in combination with zinc ion, illustrates compound H2Zinc ion in dedpa and metal β-lactamase, which combines, leads
It causes the enzyme to inactivate, there is invertibity.
Specific embodiment
Present invention will be further explained below with reference to specific examples.These embodiments are merely to illustrate the present invention and do not have to
In limitation the scope of protection of present invention.
1 compound H of application examples2Dedpa in-vitro antibacterial drug combination active testing:
The micro broth dilution method of experimental method:
(1) antibacterials storage liquid preparation: the concentration of preparation antibacterials stock solution is 5120 μ g/mL, and solubility is low
Antibacterials can be slightly less than above-mentioned concentration.Required antibacterials amount of solution or pulvis amount can formula calculated.It is prepared anti-
Bacterium medicine storage liquid should be stored in -20 DEG C or less environment, and storage life is no more than 6 months.
(2) preparation of bacterium to be measured: the single bacterium on MH (A) culture dish being incubated overnight with oese picking falls within MH (B) training
It supports in base, is calibrated to 0.5 Maxwell than turbid standard, containing about bacterium number 1 × 108Then CFU/mL dilutes 100 times to get to containing about bacterium
Number 1.0 × 106The bacterium solution of CFU/mL, it is spare.
(3) antibacterials (Meropenem MEM) stock solution mother liquor (5120 μ g/mL) is diluted 160 times respectively, obtained dense
Degree is the antibacterials solution of 32 μ g/mL.Taking 96 sterile orifice plates, the first hole is added the antibacterials of 200 μ L, and the second to ten
Hole is separately added into the MH broth bouillon of 100 μ L, draws 100 μ L from the first hole and the second hole is added, mix, then draw 100 μ L extremely
Third hole, and so on, the tenth hole is drawn 100 μ L and is discarded.Each hole drug concentration is successively at this time are as follows: 16,8,4,2,1,0.5,
0.25,200 μ L MH (B) culture mediums (negative control) are added in 0.125,0.0625,0.03125 μ g/mL, the 12nd hole.
(4) then the bacterium solution diluted is sub-packed in EP pipe, calculates concentration (the 64 μ g/ for needing fixed untested compound
ML or 32 μ g/mL), it is added into bacterium solution, makes final concentration of 64 or 32 μ g/mL of untested compound.It is trained from Metro has been diluted
1st hole of 96 orifice plates in south is separately added into the mixed solution of bacterium solution and untested compound to the 10th hole.96 orifice plates of medicine will be added
It places 37 DEG C of incubators to be cultivated, 16-18h observes bacterium solution growing state, according to U.S. clinical and laboratory standards institute
(CLSI) criterion, that hole of the completely not long bacterium of naked-eye observation are the MIC of antibacterials.Matter is done with type strain simultaneously
Control.
Experimental result is shown in Table 1, table 2.
Table 1: the compounds of this invention H2Dedpa and NOTA is trained to the MIC (μ g/mL) of the bacterial strain containing NDM and its with Metro
The MIC result of southern (MEM) drug combination
aNOTA is 1,4,7-triazacyclononane-1,4,7-triacetic acid (tri- azacyclo- nonyl of 1,4,7-
Alkane-N, N', N "-triacetic acid), NDM is New Delhi-Metallo (New Delhi metalloenzyme).
Seen from table 1, the compounds of this invention H2Dedpa can be such that all bacterial strains for carrying NDM-1 and NDM-4 enzyme restore to carbon
The sensibility of penems Meropenem.For the escherichia coli 14-55 for producing NDM-4 enzyme, and the compounds of this invention
H2MEM activity can be improved 40960 times by dedpa.
Table 2: the compounds of this invention H2Dedpa and NOTA is to the MIC (μ g/mL) and its and Meropenem for producing IMP enzyme bacterial strain
(MEM) result of drug combination
IMP is IMP type metal beta lactamase.
As can be seen from Table 2, to 10 plants of enterobacteriaceae lactobacteriaceaes of the production IMP enzyme being clinically separated, compound H2Dedpa can be with
It improves and produces IMP enzyme persister to the sensibility of Meropenem.Wherein for production IMP enzyme escherichia coli Ec-15-101, compound
The activity of MEM can be improved 32 times by NOTA, and compound H2The activity of Meropenem can be improved 64 times by dedpa.
2 compound H of application examples2The inhibiting rate and IC of the external NDM-1 enzyme of dedpa50Test:
Reaction carries out in 96 orifice plates, and final volume is 200 μ L.3 secondary orifices are arranged in parallel in each concentration of substrate, are respectively set
Experimental comparison group without Meropenem and without NDM-1 enzyme.
(1) final concentration of each substance is respectively as follows: NDM-1 3.5U, Meropenem 125mM, 10mM in reaction system
HEPES (PH=7.5), drug concentration to be measured are 10mg/mL.
The wherein every 98 μ L of hole of enzyme NDM-1 and inhibitor DMSO gradient dilution, make concentration from 10mg/mL doubling dilution to
0.15625mg/mL, every 2 μ L of hole are added in corresponding aperture, make 100 μ g/mL of final compound concentration to 1.5625 μ g/mL, use
For DMSO as negative control group, every group four parallel.It is incubated for 15min under the conditions of 30 DEG C, 100 μ L of Meropenem is added and beats
Uniformly starting reaction.
(2) 96 orifice plates are placed in microplate reader in measuring the light absorption value in its corresponding aperture at 300nm；It is measured every 60s
Once, METHOD FOR CONTINUOUS DETERMINATION 60 circulations.The enzyme inhibition rate under each inhibition concentration is calculated separately, is analyzed with Graphpad Prism5 each
The IC of inhibitor50。
With the above-mentioned active inhibiting rate of model monitoring esting sample enzyme, wherein negative control group be without inhibitor, can
With the maximum vigor of enzyme in reaction system.Blank control group is that enzyme is also free of without inhibitor, and measured result is system sheet
Floors.Therefore the calculation formula of sample to be tested inhibiting rate is as follows:
IR (%)=(1-VS/VN) × 100%
VSRepresent the enzyme reaction speed in sample to be tested hole
VNRepresent average every reaction rate of negative control hole
Experimental result is shown in Table 3.
3 compound H of table2IC of the dedpa for NDM-1 enzyme50And inhibiting rate
| Compound | IC50(μg/mL) | IC50(μM) | Inhibiting ratea(%) |
| H2dedpa | 0.15 | 0.45 | 92% |
| EDTA | 0.803 | 2.84 | 85% |
aCompound concentration is 10 μ g/mL
As shown in Table 3, compound H2Dedpa is substantially better than EDTA, compound H for the inhibitory activity of NDM-12dedpa
IC50Reach 0.45 μM.When concentration is 10 μ g/mL, its inhibiting rate reaches 92%.Higher than positive control EDTA.
3 compound H of application examples2The experiment of dedpa erythrocyte in vitro hemolytic
(1) experimental material: 10mLEP pipe, 96 orifice plates, fresh degreasing sheep blood.
(2) PBS buffer solution: 500mL specification, sodium chloride 4g, potassium chloride 100mg, sodium dihydrogen phosphate dihydrate 1.49g, nothing
Water potassium dihydrogen phosphate 100mg, deionized water are settled to 490mL, adjust between PH7.2-7.4, and sterilizing is sterilized with 10mL
Ultrapure water is added in solution after dissolving 900mg glucose.
The preparation of (3) 5% red blood cell suspensions: fresh de- fiber sheep blood is frozen in refrigerator, and configured PBS is slow
Fliud flushing is placed in 37 DEG C of water-baths, instant preparation:
It takes two 10mL EP pipes to be placed in rack for test tube, 37 DEG C of 1 × PBS is taken out into the fresh sheep blood of water-baths and refrigeration together
Alcohol is sprayed, super-clean bench is put into.1 × the PBS for drawing 5700 μ L respectively with liquid-transfering gun is added in two EP pipes, then draws 300 respectively
Microlitre sheep blood, is slowly added into PBS solution, lid lid is slowly mixed by inversion up and down, is put into 1500 turns of centrifuge and is centrifuged
10min takes out EP pipe, carefully draws supernatant, removes supernatant.It is separately added into 5~7mL PBS solution again again, slowly runs up and down
It mixes, is put into centrifugation 1500 and leaves 10min.It operates repeatedly, until centrifuged supernatant is no longer muddy.Last time from
After the heart, supernatant is skimmed, red blood cell deposit indwelling is stand-by.
It takes several 10mL EP to manage, on holding test tubes frame, 1 × PBS (37 DEG C) of 5700 μ L is added in every EP pipe, so
The red blood cell deposit of 300 μ L is sequentially added afterwards.It is slowly mixed by inversion up and down, in this way, the just red cell suspension of configuration 5%.
(4) configuration of sample solution: (DMSO final concentration cannot be greater than 0.5%) is dissolved with a small amount of DMSO, and uses phase
The DMSO of same volume does negative control.Dissolved DMSO with PBS dilute (for example, the first hole concentration be 1000 μ g/mL, then
The content of drug is exactly 2mg in the 50 μ L that first hole is added, and is configured to the solution of 2mg/50 μ L), the medicine in this branch EP pipe at this time
Object is initial drug.Then it takes nine 1.5mL EP pipes to be placed in rack for test tube in parallel, is separately added into the PBS (number 2 of 200 μ L
Number, No. 3, No. 4 ... No. 10).The all such operation repetitive of all drugs.Finally, by drawing 200 μ L's in initial drug EP pipe
Drug solution is added in No. 2 EP pipes, draws in 200 μ L to No. 3 EP pipes after purging repeatedly, purges ... repetitive operation repeatedly, directly
It is managed to No. 10 EP.Drug is so diluted.
(5) bed board: taking 96 orifice plates, finishes writing experiment numbers, drug code, date.Liquid-transfering gun is adjusted to 150 μ L, will be configured
It is light and slow above and below 5% good red cell suspension to be mixed by inversion, it successively draws and spreads in 96 orifice plates (6 × 10).It then will be configured
Drug is corresponding to be added in 96 orifice plates, three multiple holes of a drug.It is placed after adding and is incubated for 1h in 37 DEG C of insulating boxs.
(6) it post-processes: 96 orifice plates is taken out out of insulating box, be placed in -4 DEG C of centrifuges and be centrifuged (3500rpm, 5min).
Centrifugation finishes, and every block of plate is corresponding all to take one piece of 96 new orifice plate.Plank control after mark and centrifugation.Then it accordingly draws
100 μ L supernatants (hole hole is corresponding).After absorption, and OD value is measured in microplate reader, analyze data, obtain HC50。
Experimental result is shown in attached drawings 1.
4 compound H of application examples2The test of dedpa vitro cytotoxicity
Experimental material: DMEM culture medium, cell counting board, 96 orifice plates, CCK-8, microplate reader.
(1) aseptic subpackaged bottle, the proportional arrangement of FBS and DMEM culture medium 1:10, in 4 DEG C of refrigerators the configuration of culture medium: are taken
Interior storage is stand-by.
(2) bed board: Hela cell in good condition in culture is taken out from incubator, is operated in Biohazard Safety Equipment.
Supernatant culture medium is outwelled, it is primary to wash cell with 2mL PBS, removes remaining culture medium.1mL trypsase is taken to squeeze into training along wall
It supports in ware, in 37 DEG C of incubators, percent by volume 0.5%CO2Middle incubation 1-2min.Postdigestive culture dish is taken out, 1mL is added
Culture medium terminates digestion.Postdigestive cell suspension will be terminated to be transferred in 10mL EP pipe, 800rpm 3min centrifugation.It takes out
EP pipe, slowly outwells supernatant, and 2-4mL culture mediums are added, and blows and beats 50 times repeatedly.10 μ L cell suspensions are therefrom taken to squeeze into cell
In tally, counted under 20 × microscope.The number for needing cell is calculated, the cell suspension in 5000/hole is configured to.
Every 100 μ L of hole is sequentially added in 96 orifice plates, is incubated for for 24 hours in constant incubator.
(3) dosing: by drug to be measured after gradient dilution is good in 4ml EP pipe, 96 orifice plates of adherent growth are taken out, are abandoned
Fall supernatant, by the drug to be measured diluted three secondary orifices of each concentration, every 200 μ L of hole is added in plate, by 96 holes after the completion of operation
Plate is put into constant incubator.After for 24 hours, the every 7 μ L of hole of cell count reagent cck-8 is sequentially added in plate, is put into constant incubator
After middle culture 4h in microplate reader, OD value is read at 450nm wavelength, is calculated inhibiting rate, is returned its IC50。
Experimental result is shown in attached drawings 2.
5 compound H of application examples2Dedpa lives in vitro, and dead cell is double to contaminate test
(1) experimental material: DMEM culture medium, cell counting board, 96 orifice plates, CCK-8, calcein-AM, propidium iodide
(PI), PBS buffer solution, fluorescence microscope.
(2) aseptic subpackaged bottle, the proportional arrangement of FBS and DMEM culture medium 1:10, in 4 DEG C of refrigerators the configuration of culture medium: are taken
Interior storage is stand-by.
(3) configuration of fluorescent dye: the calcein-AM dispensed and propidium iodide (PI) are diluted with PBS, instant
Dilution.
(4) bed board: Hela cell in good condition in culture is taken out from incubator, is operated in Biohazard Safety Equipment.
Supernatant culture medium is outwelled, it is primary to wash cell with 2mL PBS, removes remaining culture medium.1mL trypsase is taken to squeeze into training along wall
It supports in ware, in 37 DEG C of incubators, percent by volume 0.5%CO2Middle incubation 1-2min.Postdigestive culture dish is taken out, is added
1mL culture medium terminates digestion.Postdigestive cell suspension will be terminated to be transferred in 10mL EP pipe, 800rpm 3min centrifugation.
EP pipe is taken out, supernatant is slowly outwelled, 2-4mL culture mediums are added, is blown and beaten 50 times repeatedly.10 μ L cell suspensions are therefrom taken to squeeze into
In cell counting board, counted under 20 × microscope.The number for needing cell is calculated, the cell for being configured to 30000/hole is mixed
Suspension.Every hole 1mL is sequentially added in 12 orifice plates, is incubated for for 24 hours in constant incubator.
(5) dosing and dyeing: by drug to be measured after gradient dilution is good in 4mL EP pipe, 12 holes of taking-up adherent growth
Plate discards supernatant, and the every 700 μ L of hole of the drug to be measured diluted is added in plate, and 12 orifice plates are put into constant temperature training after the completion of operation
It supports in case.It after for 24 hours, takes out, collects the supernatant in each hole respectively, mark, 3500rpm 5min is centrifuged supernatant, discards supernatant
Dead cell is collected, is then washed once with PBS, discards PBS.12 orifice plates are washed once with the every hole PBS.It is every with configured dyestuff
700 μ L of hole first blows and beats the dead cell for collecting the hole, is then mixed liquid and is added in the hole of 12 orifice plates.After the completion of operation, in
Constant incubator is incubated for 15min.Then, it takes pictures under Nikon fluorescence microscope.
Experimental result is shown in attached drawings 3.
6 compound H of application examples2Dedpa extracorporeal disinfecting kinetic test
(1) experimental material: MHB culture medium (Muiller-Hinton Broth), MHA culture medium (Muiller-Hinton
Agar), 1.5mL sterile EP tube, 96 orifice plates, PBS buffer solution.
(2) configuration of culture medium: a certain amount of MHA and MHB powder, 121 DEG C of high-temperature sterilization 20min are dissolved with ultrapure water
Afterwards, it takes out spare.MHA culture medium waits for that it is cooled to 40 DEG C or so and is poured into disposable sterilized culture dish, to condense to room temperature
At solid medium.
(3) it step: takes two 10mLEP pipes to be separately added into the MHB culture medium of 3mL, is picked them separately in culture dish with oese
Monoclonal bacterial strain (NDM-4 bacterial strain 14-55 and IMP bacterial strain Ec-101), in constant-temperature table, 37 DEG C of 250rpm overnight growths.
Second day, the bacterium solution in two branch pipes is all diluted by 1:10000 times respectively, dilutes 14 respectively and manage, the bacterium solution of every pipe 3mL,
It is divided into two batches, grows 2h and 5h respectively at 37 DEG C of 250rpm.Growth time to be reached takes out bacterium solution, and the anti-of setting concentration is added
Bacterium drug is centrifuged at this point, taking out the sample liquid of 100 μ L inside each sample cell respectively in 10000g in refrigerated centrifuge, is abandoned
Fall supernatant, is diluted with 1 × PBS.Sterile 96 orifice plate is taken, 1 × PBS is added in every 180 μ L of hole.It has been diluted from above-mentioned with 1 × PBS
20 μ L are taken out in bacterium solution, the first hole is added, then doubling dilution backward.Taken out from the hole diluted the sample drop of 10 μ L to
It on MHA solid medium, marks, bacterium colony when this is 0h counts.Backward according to the above method, successively to 1h, 2h,
3h ... is equal for 24 hours to carry out bacterium colony counting, draws curve.The results are shown in attached figure 4.
7 compound H of application examples2The external zinc ion drug combination experiment of dedpa
(1) antibacterials storage liquid preparation: the concentration of preparation antibacterials stock solution is 5120 μ g/mL, and solubility is low
Antibacterials can be slightly less than above-mentioned concentration.Required antibacterials amount of solution or pulvis amount can formula calculated.It is prepared anti-
Bacterium medicine storage liquid should be stored in -20 DEG C or less environment, and storage life is no more than 6 months.
(2) preparation of bacterium to be measured: the single bacterium on MH (A) culture dish being incubated overnight with oese picking falls within MH (B) training
It supports in base, is calibrated to 0.5 Maxwell than turbid standard, containing about bacterium number 1 × 108Then CFU/mL dilutes 100 times to get to containing about bacterium
Number 1.0 × 106The bacterium solution of CFU/mL.
(3) antibacterials (Meropenem) stock solution mother liquor (5120 μ g/mL) is diluted 160 times respectively, obtaining concentration is
The antibacterials solution of 32 μ g/mL.96 sterile orifice plates are taken, the antibacterials of 200 μ L, the second to ten hole difference is added in the first hole
The MH broth bouillon of 100 μ L is added, draws 100 μ L from the first hole and the second hole is added, mix, then draw 100 μ L to third
Hole, and so on, the tenth hole is drawn 100 μ L and is discarded.Each hole drug concentration is successively at this time are as follows: 16,8,4,2,1,0.5,0.25,
0.125,200mL MH (B) culture medium (negative control) is added in 0.0625,0.03125 μ g/mL, the 12nd hole.
(4) then the bacterium solution diluted is sub-packed in EP pipe, calculates concentration (the 64 μ g/ for needing fixed untested compound
ML or 32 μ g/mL), it is added into bacterium solution, makes final concentration of 64 or 32 μ g/mL of untested compound, equimolar is then added
The ZnCl of amount2.The mixed of bacterium solution and untested compound is separately added into from the 1st hole to the 10th hole for 96 orifice plates for having diluted Meropenem
Close solution.96 orifice plates for adding medicine, 37 DEG C of incubators of placement are cultivated, 16-18h observes bacterium solution growing state, according to the U.S.
The clinical criterion with laboratory standards institute (CLSI), the hole of the completely not long bacterium of naked-eye observation are the MIC of antibacterials.
Quality Control is done with reference culture escherichia coli ATCC 25922 simultaneously.The results are shown in attached figure 5.
By above-mentioned Bioactivity evaluation experimental it is found that compound H2Dedpa have can make drug resistant Meropenem
Restore its sensitive bioactivity, the activity of Meropenem can be improved 40960 times by highest.In vitro toxicity is it is experimentally confirmed that red
Cytotoxicity shows compound H2Even if dedpa is when concentration is 1000 μ g/mL for red blood cell still without what toxicity.And
The toxicity of external Hela cell is shown, compound H2Dedpa is for vitro cytotoxicity also in its therapeutic dose MIC range
Within.It sterilizes dynamic (dynamical) the result shows that compound H2Dedpa there is sterilization well to live the bacterium at logarithmic growth initial stage
Property.The Inhibition test of NDM-1 enzyme also demonstrates compound H2Dedpa can inhibit the activity of NDM-1 really, and to NDM-1
The IC of enzyme50Less than EDTA.Zinc ion drug combination experimental verification compound H2The zinc ion of dedpa and metallo-β-lactamase
In conjunction with causing enzyme to inactivate.A series of biological evaluation can tentatively assert, compound H2Dedpa has potential joint patent medicine
Value.
Claims (2)
1. open chain pyridine carboxylic acid derivatives H2Application of the combination of dedpa and Meropenem in preparation antibacterials, the compound
Structural formula is as follows, which is characterized in that it is as metallo-β-lactamase inhibitor, and Meropenem drug combination, for antibacterial,
Sterilization, improves antibody-resistant bacterium to the sensibility of Meropenem,
。
2. open chain pyridine carboxylic acid derivatives H as described in claim 12The combination of dedpa and Meropenem is in preparation antibacterials
In application, which is characterized in that the antibody-resistant bacterium be escherichia coli, C. freundii, enterobacter cloacae, perfringens
Clostruidium, Friedlander's bacillus, proteus mirabilis.
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| WO2016030104A1 (en) * | 2014-08-29 | 2016-03-03 | Anmi S.A. | Mono-, di- or polysaccharide used as metal inhibitor in the preparation of 68ga-chelate-functionalized targeting agent |
| WO2016046793A2 (en) * | 2014-09-26 | 2016-03-31 | The South African Nuclear Energy Corporation Limited | Radiopharmaceutical conjugate |
| WO2016054277A1 (en) * | 2014-09-30 | 2016-04-07 | University Of Pittsburgh -Of The Commonwealth System Of Higher Education | Cu(i)-catalyzed azide-alkyne cycloadditions (cuaac) ligands and methods for carrying out cu(i)-catalyzed azide- alkyne cycloaddition reactions |
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| WO2016030104A1 (en) * | 2014-08-29 | 2016-03-03 | Anmi S.A. | Mono-, di- or polysaccharide used as metal inhibitor in the preparation of 68ga-chelate-functionalized targeting agent |
| WO2016046793A2 (en) * | 2014-09-26 | 2016-03-31 | The South African Nuclear Energy Corporation Limited | Radiopharmaceutical conjugate |
| WO2016054277A1 (en) * | 2014-09-30 | 2016-04-07 | University Of Pittsburgh -Of The Commonwealth System Of Higher Education | Cu(i)-catalyzed azide-alkyne cycloadditions (cuaac) ligands and methods for carrying out cu(i)-catalyzed azide- alkyne cycloaddition reactions |
Non-Patent Citations (1)
| Title |
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| Evaluation of the h2dedpa scaffold and its crgdyk conjugates for labeling with 64Cu;Eszter Boros等;《Inorganic Chemistry》;20120514;第51卷;6279-6284 * |
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