CN103951680A - Application of novel metal beta-lactamase inhibitor in preparation of medicines for resisting drug-resistance bacteria - Google Patents
Application of novel metal beta-lactamase inhibitor in preparation of medicines for resisting drug-resistance bacteria Download PDFInfo
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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
- A61K31/41—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
- A61K31/425—Thiazoles
- A61K31/429—Thiazoles condensed with heterocyclic ring systems
- A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
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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
- A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
- A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
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Abstract
The invention discloses a NDM-1 metal beta-lactamase inhibitor. The NDM-1 metal beta-lactamase inhibitor is capable of inhibiting the inactivation of beta-lactam antibiotics and restoring the antibacterial activity of the beta-lactam antibiotics. A novel compound with the structure as shown in a general formula (I) has the inhibitory activity of NDM-1 metal beta-lactamase. The invention also discloses an application of a composition of the compound (I) and the beta-lactam antibiotics in restoration of antibacterial activity of the beta-lactam antibiotics on bacteria capable of generating the NDM-1 metal beta-lactamase inhibitor.
Description
Technical field
The present invention relates to a kind of novel cpd as NDM-1 metallo-β-lactamase inhibitor, and more particularly the present invention relates to pharmaceutical composition, this pharmaceutical composition and beta-lactam antibiotics combine to improve the bacterium infection that resists the effect of the tolerant bacteria that produces NDM-1 metallo-β-lactamase and be used for the treatment of the mankind.
Background technology
Nowadays, microbiotic has become the indispensable class medicine of the mankind, but abuse of antibiotics makes bacterium obtain multi-drug resistant from different levels and mode, and wherein Gram-negative bacteria secretion β-lactamase becomes the mechanism that bacterial drug resistance is the most general already.β-lactamase can be divided into A according to the difference of its molecular structure, B, and C and D tetra-classes (Ambler classification), or according to its substrate, inhibitor and molecular structure classify (Bush-Jacoby labelling method).According to Ambler classification, A, C, D class is Serine β-lactamase, and its avtive spot is a serine residue, and the hydroxyl on this serine residue can be used as the carbonyl on nucleophilic reagent attack beta-lactam nucleus, thereby microbiotic was lost efficacy.At present, be widely used clinically for example clavulanic acid for the inhibitor of part Serine β-lactamase.But this class inhibitor is but completely invalid to an other class β-lactamase, i.e. category-B β-lactamase.Category-B β-lactamase claims again metallo-β-lactamase (metallo β-lactamases, MBLs), and this fermentoid needs one or two divalent metal to assist its performance catalytic activity conventionally.MBLs can catalytic hydrolysis in single most β-lactam antibiticss acyl lopps, simultaneously can also the unhydrolyzable carbapenem antibiotic of catalytic hydrolysis Serine β-lactamase.MBLs can be divided into B1 according to the specificity of the metal ion of the similarity of its aminoacid sequence, combination and substrate again, B2, tri-subclass of B3.B1 and B3 subclass have two zine ion binding sites, can the most of β-lactam antibitics of catalytic hydrolysis including cephalosporins, carbapenems and penicillins.B2 subclass only has a zine ion binding site, cephalosporins and Penicillin antibiotics is shown to weak hydrolytic activity, and carbapenems is shown to very high catalytic hydrolysis activity.Although the hydrolysis mechanism of different subclass is different, the metal ion of avtive spot is all showing important effect in the time that microbiotic is combined, and can produce a kind of open loop of nucleophile promotion beta-lactam nucleus, thereby causes antibiotic inefficacy.
In December, 2009, from the Sweden patient of 1 routine pneumonia infection klebsiella, be separated to a kind of novel metal β-lactamase, be New Delhi metallo-β-lactamase (New Delhi metallo-β-lactamase-1, NDM-1), this kind of enzyme can be hydrolyzed nearly all β-lactam antibitics, comprise the most effective carbapenems at present, brought great threat to clinical treatment.Subsequently the U.S., Britain, Belgium, India, sent out, Klebsiella Pneumoniae that Japan, Hong-Kong, TaiWan, China, Australia have found respectively to carry NDM-1 gene, escherichia coli, enterobacter cloacae, Acinetobacter bauamnnii, citric acid bacillus etc.Nearly 200 kinds of microbiotic of whole world listing are almost felt simply helpless to this Novel super bacterium.At present, only have two kinds of microbiotic of polymyxin and Tigecycline can suppress to a certain extent wreaking havoc of NDM-1.Due to the diversity of the fast propagation of NDM-1, hydrolysis substrate and possess the ability of catalytic hydrolysis carbapenem antibiotic, still do not have clinically effective inhibitor to occur, therefore the exploitation of NDM-1 metallo-β-lactamase inhibitor is extremely urgent.
Summary of the invention
The object of this invention is to provide new metallo-β-lactamase inhibitor, it is used for suppressing the deactivation of beta-lactam antibiotics and recovers anti-microbial activity as medicine.
Present inventor has been found that compound or its salt or the solvate of formula (I) expression is inhibited to NDM-1 metallo-β-lactamase now.In addition, have been found that compound or its salt that formula (I) represents or solvate can be effective to recover beta-lactam antibiotics antagonism and produce the activity of the bacterium of NDM-1 metallo-β-lactamase.The present invention is based on these discoveries.
The invention provides NDM-1 metallo-β-lactamase inhibitor, it comprises the compound or its salt or the solvate that are represented by following formula (I):
Wherein
R
1represent hydrogen atom or C
1-6alkyl, all these groups can be substituted,
R
2represent hydrogen atom, C
1-8alkyl, C
3-8cycloalkyl, aryl, iso-aryl, phenyl-C
1-6alkyl, phenylamino acyl group, C
3-8cycloalkyl-C
1-6alkyl, iso-aryl-C
1-6alkyl, all these groups can be substituted,
R
3represent hydrogen atom, cyano group, nitro, halogen atom, C
1-6alkyl, C
1-6haloalkyl, C
1-6hydroxyalkyl, C
1-6aminoacyl, C
3-7cycloalkyl, aryl, iso-aryl, different cycloalkyl, hydroxyl, carbonyl, ester group, amino, all these groups can be substituted.
Unless otherwise indicated, term " C
1-6", " C
1-8", " C
2-8", " C
3-7", " C
3-8" etc. represent carbon atom number, and for example " C
1-6alkyl " represent to have the alkyl of 1-6 carbon atom.Rudimentary is preferably C
1-6, and in the situation of cyclic group, it preferably represents C
3-7.
Term " halogen atom " represents fluorine atom, chlorine atom, bromine atoms or iodine atom.
Term " alkyl " preferably represents to have the straight or branched alkyl of 1-6 carbon atom as the part of group or group.The example of " alkyl " comprises methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, sec-butyl, the tertiary butyl, n-pentyl, neo-pentyl, isopentyl, tert-pentyl, n-hexyl, isohexyl etc.
Term " cycloalkyl " preferably represents to have the monocycle alkyl of 3-7 carbon atom as the part of group or group.The example of " cycloalkyl " comprises cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, suberyl etc.In addition, low-grade cycloalkyl preferably represents C defined above
3-7cycloalkyl.
Term " aryl " preferably represents aromatic ring, the such as phenyl of phenyl, replacement etc., and thick and ring, such as naphthyl, phenanthryl, fluorenyl, anthryl etc.Preferred aryl comprises phenyl, naphthyl and fluorenyl.
R
1term " the C representing
1-6alkyl " can be straight or branched, preferably C
1-3alkyl, such as methyl, ethyl, n-propyl, sec.-propyl etc., more preferably methyl, ethyl.Alkyl can be substituted, and the preferred hydroxyl of substituting group, amino and halogen atom.
R
2the term " phenylamino acyl group " representing can be following several structure:
R
3the term " halogen atom " representing comprises above-mentioned " halogen atom ", comprises fluorine atom, chlorine atom, bromine atoms or iodine atom, and preferred chlorine atom or bromine atoms.
Preferably pharmacologically acceptable salt of general formula (I) compound, it comprises acid salt.Therefore, general formula (I) compound can be with the form application of the salt derived from mineral acid or organic salt, described salt comprises acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, hydrosulfate, butyrates, Citrate trianion, camphorate, camsilate, cyclopentane propionate, digluconate, dodecyl sulfate, esilate, fumarate, glucoheptose salt, glycerophosphate, Hemisulphate, enanthate, hexanoate, hydrochloride, hydrobromate, hydriodate, 2-isethionate, lactic acid salt, maleate, mesylate, 2-naphthalenesulfonate, nicotinate, oxalate, embonate, pectate, persulphate, 3-phenylpropionic acid salt, picrate, Pivalate, propionic salt, succinate, tartrate, thiocyanate-, tosylate and undecane hydrochlorate.
General formula (I) compound can be the form of solvate.
The solvent of solvate comprises water, methyl alcohol, ethanol, Virahol, butanols, acetone, ethyl acetate, chloroform, dimethyl sulfoxide (DMSO) etc.
General formula (I) compound or its salt can comprise asymmetric carbon in molecule, and each isomer or all isomer mixtures comprise in the present invention.In addition, general formula (I) compound can be applied with prodrug forms.Prodrug can be hydrolyzed and therefore have precedence over Orally administered in vivo, and this is absorption, the acidproof and other factors having had from stomach mucous membrane or intestinal mucosa due to it.
Technical solution of the present invention is specific as follows:
(1) expression of NDM-1 and separation and purification, obtains NDM-1 metallo-β-lactamase of the present invention;
(2) the NDM-1 inhibitor virtual screening based on molecular docking, screens formula of the present invention (I) compound;
(3) body outer suppressioning test of formula of the present invention (I) compound to NDM-1.
Above-mentioned expression system is selected prokaryotic expression system, the preferred escherichia expression system of prokaryotic expression system, and expression system is generally adapted to the expression of recombinant protein of the present invention, the preferred pET28a of expression vector in wherein said intestinal bacteria system.
A kind of preferred version of above-mentioned Expression and purification method is: the gene transformation of the coding NDM-1 recombinant protein having built is arrived to e. coli bl21 (DE3), obtain through low temperature induction, liquid culture engineering bacteria the target protein of secreting soluble form to born of the same parents, centrifugal collection thalline, ultrasonication cell, the centrifugal supernatant that obtains having NDM-1 recombinant protein.Utilize the method for Ni-NTA resin affinity chromatography to carry out purifying, purity reaches more than 90%, obtains recombinant protein of the present invention.
Above-mentioned virtual screening method is selected the Discovery Studio v2.5.5 of Accelrys company exploitation, it uses Libdock technology to realize docking of one group of small molecules and acceptor, the micromolecular compound of the preferred ZINC database of wherein said small molecules, the crystalline structure (PDB ID:3Q6X) of the preferred NDM-1 metallo-β-lactamase of described acceptor and Ampicillin Trihydrate hydrolysate.Obtain formula of the present invention (I) compound through molecular docking, marking sequence etc.
Above-mentioned body outer suppressioning test relates generally to the inhibition of formula of the present invention (I) compound to NDM-1 metallo-β-lactamase activity and inhibiting rate, IC
50mensuration; Also relate to formula of the present invention (I) compound and beta-lactam antibiotics uses simultaneously, to producing the inhibition of bacterial growth of NDM-1 metallo-β-lactamase, the preferred Ampicillin Trihydrate of wherein said beta-lactam antibiotics, the described bacterium that can the produce NDM-1 metallo-β-lactamase NDM-1 intestinal bacteria of preferably recombinating.
Brief description of the drawings
Fig. 1 is the SDS-PAGE electrophorogram of NDM-1 metallo-β-lactamase Expression and purification: wherein swimming lane 1 is Marker; Swimming lane 2 is the front full bacterium of induction; Swimming lane 3 is the rear full bacterium of induction; Swimming lane 4 is supernatant after broken bacterium; Swimming lane 5 is broken bacterium postprecipitation; Swimming lane 6 is worn liquid for upper clear stream; Swimming lane 7 is 200mM imidazoles elutriant; Swimming lane 8 is albumen after ultrafiltration and concentration.
Fig. 2 is extracorporeal bacteria inhibitor test disk diffusion method result: wherein A is NDM-1-pET28aE.coli; B is pET28aE.coli; Disc1:Amp2.5 μ g; Disc2:Amp2.5 μ g+compound20nmol; Disc3:Amp2.5 μ g+compound40nmol; Disc4:Amp2.5 μ g+compound60nmol; Disc5:Amp2.5 μ g+EDTA60nmol; Disc6:Amp2.5 μ g+L-Cap60nmol; Disc7:compound60nmol.
Specific implementation method
Below in conjunction with drawings and Examples, the invention will be further described.
Expression and the separation and purification of embodiment 1:NDM-1 metallo-β-lactamase
(1) conversion of NDM-1 recombinant plasmid: get the correct plasmid of sequence and joint 1 μ L and add in 20 μ L BL21 competent cells, ice bath 30min, 42 DEG C of heat shock 90s, then ice bath 3min.Add 500 μ L LB liquid nutrient mediums, 37 DEG C, 180rpm shaking culture 45-60min.The centrifugal 3min of EP pipe 3000rpm, draws the centrifugal supernatant of 400 μ L, will after thalline suspendible, use spreading rod to be evenly applied to Kan
ron flat board.Face up and be placed in 37 DEG C of constant incubators, after thalline is absorbed by substratum, be inverted overnight incubation.Some single bacterium colony in the above-mentioned flat board of picking, is inoculated into (Kan in 5mL LB liquid nutrient medium
rresistance, working concentration 50 μ g/mL), 37 DEG C, 220rpm shaking culture is spent the night, after add 15% glycerine and preserve some parts of bacterial classifications in-80 DEG C.
(2) abduction delivering of NDM-1 albumen: expression bacterium E.coli BL21 (pET28a:NDM-1) the 50 μ L that get-80 DEG C of preservations are inoculated in 50mL containing in the LB liquid nutrient medium of kantlex (working concentration 50 μ g/mL), 37 DEG C, 220rpm shaken overnight is cultivated, and obtains seed liquor; With the ratio of 1: 100,2mL seed liquor is transferred to 200mL containing in the LB liquid nutrient medium of kantlex, 37 DEG C, 220rpm shaking culture; As the OD of bacterium liquid
600value is about at 0.8 o'clock, gets 1mL bacterium liquid, and 4 DEG C, the centrifugal 2min of 12000rpm, collects bacterial sediment, and be cooled to 20 DEG C, adds 200 μ L1M IPTG liquid storages (working concentration 1mM), with the speed oscillation induction 8h of 180rpm; Get the rear bacterium liquid of 1mL induction, 4 DEG C, the centrifugal 2min of 12000rpm, collects bacterial sediment, and all the other bacterium liquid pack in 200mL Centrifuge Cup, and 4 DEG C, the centrifugal 20min of 4500rpm, abandons supernatant, weighs thalline weight, frozen in-80 DEG C.
(3) acquisition of NDM-1 albumen supernatant: get the thalline of-80 DEG C of preservations, add Bindingbuffer (pH7.4) resuspended evenly according to the ratio of 10mL/g, add proteinase inhibitor PMSF to final concentration be 1mM.Subsequently bacterium liquid is placed in to glass beaker, intermittently 3s of the ultrasonic 3s of 400W under condition of ice bath, each ultrasonic 15min, limpid to bacterium liquid till.After bacterial cell disruption, 4 DEG C, the centrifugal 30min of 12000rpm, collects respectively upper cleer and peaceful precipitation.Precipitation is resuspended with the isopyknic PBS solution of supernatant.
(4) purifying of NDM-1 albumen: Ni-NTA dress post, column volume is 2mL left and right; With 5 column volumes of Binding buffer (pH7.4) balance, flow velocity is 2mL/min; Supernatant is crossed to 0.45 μ m membrane filtration, after load on Ni affinity column, flow velocity is 1mL/min; In collection, clear stream is worn sample, by SDS-PAGE electrophoresis detection hanging column situation; Wash 5 column volumes with Binding buffer (pH7.4), flow velocity is 2mL/min again; With respectively containing 50,100,200, the Binding buffer (pH7.4) of 500mM imidazoles carries out gradient elution, flow velocity is 1mL/min; Collect each gradient elution sample, by the purifying situation of SDS-PAGE electrophoresis detection SIM-1 albumen; Wash 5 column volumes with pure water, then wash 5 column volumes with 20% ethanol, flow velocity is 2mL/min, finally uses 4 DEG C of preservation post materials of 20% ethanol.
(5) ultrafiltration and concentration of NDM-1 albumen: get the 200mM imidazoles elutriant that purity is the highest, pour in Millipore ultrafiltration and concentration pipe; 4 DEG C, the each centrifugal 15min of 4500 × g, is less than 1mL to liquid volume in evaporating pipe; To adding the not Binding buffer (pH7.4) containing imidazoles in evaporating pipe, continue centrifugal according to above-mentioned parameter, repeated multiple times, until imidazole concentration goes to zero in evaporating pipe; Get the protein solution finally having concentrated, every pipe 100 μ L divide and install in low absorption EP pipe, in-80 DEG C of preservations.
(6) SDS-PAGE experimental implementation flow process is as follows: get before and after induction, after broken bacterium, supernatant, precipitation and above-mentioned purifying, the concentrated rear each 40 μ L of sample that collect, add 10 μ L5 × SPS-PAGE sample-loading buffers, boiling water bath 5min; Record SDS-PAGE glue with reference to " molecular cloning handbook ", 5% concentrated glue, 12% separation gel, 1 × Tri-Gly protein electrophoresis damping fluid; Respectively get 10 μ L supernatant liquor loadings, carry out electrophoresis, parameter is the about 1h of 150V after the about 20min of constant voltage 90V; After finishing, takes off electrophoresis SDS-PAGE glue, coomassie brilliant blue staining 1-2h, then decolour with destainer.Protein electrophoresis the results are shown in Figure shown in 1.The above albumen solubility expression in e. coli bl21 (DE3) as seen from the figure, and after purifying, obtain the NDM-1 recombinant protein that purity is greater than 90%.
Embodiment 2: the NDM-1 inhibitor virtual screening based on molecular docking
(1) definition albumen is acceptor molecule: in Files browser, find data file 3Q6X.pdb, mouse is double-clicked, and this albumen will occur in a new three dimensional window.Click and select drop-down menu Chemistry|Hydrogens|Add Polar to add polarity hydrogen to albumen.In system view, launch <Cell>, click and select 3Q6X.And then in Tools browser, from drop-down list, select Receptor-Ligand Interactions, open Receptor-Ligand Interactions object palette, in Define set of tools under Define and Edit Binding Site object palette, click " Define Selected Molecule as Receptor " the protein molecular 3Q6X selecting is above defined as to acceptor molecule, for next step.
(2) find possible binding site in acceptor: in Define object palette, click " Find Sites from Receptor Cavities " and find possible binding site in acceptor.
(3) from binding site definition sphere ball: click system view and select binding site, the Transform-Fit To Screen button of clicking in View toolbar amplifies binding site, then click the Define Sphere from Selection in Define set of tools under Define and Edit Binding Site object palette, this defines a red ball at combining site, and the name of ball is called SBD_Site_Sphere.
(4) in system view, click and select " SBD_Site_Sphere ", then a mouse click right button is selected " Attributes of SBD_Site_Sphere ", open " SphereObject Attributes " dialog box, in dialog box, find that a line of radius (Radius), value is set to 9, hit OK button.
(5) open part file: in file browser, find part file, double-click, part is opened in new window.
(6) open flow process, amendment parameter: in Protocols browser, launch Receptor-Ligand Interactions folder, double-click Dock Ligands (LibDock) flow process, the corresponding parameter of flow process is opened in parameter browser.In parameter browser, click Input Receptor parameter, from drop-down list, select " 3Q6X:3Q6X " that receptor protein is set.Click Input Ligand parameter, from drop-down list, select all parts, specify docking part.Click Input Site Sphere parameter, from drop-down list, select the coordinate of sphere.Click Docking Preferences parameter, select " User Specified " from drop-down list, this allows for docking and calculates the specific parameter of change.Launch Docking Preference parameter, click Max Hits to Save parameter, input value " 10 ".
(7) operation molecular docking is calculated, check result: click the start button running job in flow process toolbar (Protocols toolbar), after waiting for that operation completes, double-click the molecular docking operation just having completed in operation browser, open Report.htm, click the View Results link of Output part in this file, open the result that docking is calculated.In the form of opening, demonstrate the part best pose that gives a mark.
(8) browse docking poses: click and activate " Molecule-Molecule Window " window, guarantee that system view and form browser open, otherwise open by CTRL+H and CTRL+T.Press CTRL+1 and hide flow process browser and operation browser.In Tools browser, from drop-down list, select Receptor-Ligand Interactions, open Receptor-Ligand Interactions object palette, under Visualize Receptor-Ligand Interactions object palette, click " Receptor-Ligand Hydrogen Bonds ", in view window, the hydrogen bond that acceptor atom docks with part between poses can show by green line.
(9) analyze docking result, generate part-protein-interacting 2 d plane picture: select drop-down menu File|New | Molecule Window, opens a new window.Click and activate docking result place " Molecule-Molecule Window " window, in system view, select albumen 3Q6X, and pin Ctrl and select first ZINC21157214 that Libdock Score is the highest simultaneously, a mouse click right button, selects " Copy " order in right-click menu.Activate the window of newly opening, click 3D Window, and a mouse click right button, " Paste " order in right-click menu, selected.This operation is placed on albumen and ZINC21157214 separately in a new window.In system view, click and choose part, pin left mouse button and be drawn in albumen 3Q6X, can see that part becomes a part for acceptor, name is " <Chain> ".In Tools browser, from drop-down list, select Receptor-Ligand Interactions, open Receptor-Ligand Interactions object palette, in Define and Select Ligands set of tools under Analyze Binding Site object palette, click " Create Ligand Groups ", can see that system view has increased one " ligand0 ".In Tools browser, click Receptor-Ligand Interactions|Analyze Binding Site|Draw Ligand Interaction Diagram|Draw Diagram order, can see and in a new window, open part-protein-interacting 2 d plane picture, be convenient to observe more intuitively both interaction and crucial amino acid and groups.
Embodiment 3: the body outer suppressioning test of formula of the present invention (I) compound to NDM-1
(1) mensuration of the extracorporeal inhibiting rate of formula of the present invention (I) compound to NDM-1: NDM-1 stock concentrations is that (system damping fluid is 30mM HEPES to 2nM, 10 μ M ZnCl
2, pH6.8), Nitrocefin stock concentrations is that (solvent is 1mMDMSO to 200 μ M, and system damping fluid is 30mM HEPES, 10 μ M ZnCl
2, pH6.8), negative control is 3.0 μ L DMSO, positive control is 3.0 μ L1mM EDTA-DMSO solution.
Experimental group is got 100 μ L NDM-1 liquid storages and is mixed with 3 μ L compound liquid storages (1mM DMSO), hatches 15min for 30 DEG C, and to ensure the complete combination of compound and NDM-1, control group does not add compound; Add subsequently 100 μ L Nitrocefin liquid storages, end reaction system is 100 μ M Nitrocefin again, 2nM NDM-1,30mM HEPES, 10 μ M ZnCl
2, pH6.8.Control group and experimental group are each to be repeated 4 times, measures the variation of absorption value at 495nm place, and every 30sec reads absorbance one time, measures altogether 15min.Inhibiting rate=Δ A
495nmexperimental group/Δ A
495nmpositive controls × 100%.Through data analysis, formula of the present invention (I) compound is 56.5 ± 2.6%, IC to the inhibiting rate of NDM-1 activity
50value is 31.4 ± 1.2 μ M.
(2) extracorporeal bacteria inhibitor test disk diffusion method: NDM-1 glycerol stock, pET28a glycerol stock are inoculated in respectively to 5mL containing in the LB substratum of Kana, 37 DEG C, 220rpm, overnight incubation; Be transferred in 100mL LB substratum by 1: 50,37 DEG C, 220rpm, to OD
600=～0.8, be cooled to 20 DEG C, 180rpm, wherein NDM-1 adds 100 μ L IPTG induction 2h; After 2h, with aqua sterilisa, bacterium liquid is diluted to OD
600=～0.13, spread upon equably on MH solid medium drying at room temperature 5min with sterilized cotton swabs rod; Drug sensitive test paper is placed on substratum with sterilizing tweezers, adds formula of the present invention (I) compound of Ampicillin Trihydrate and/or different concns, after incubated at room 15min, be inverted in incubated overnight in 37 DEG C of incubators.
As seen in Figure 2, dull and stereotyped A is NDM-1 intestinal bacteria to result, and dull and stereotyped B is pET28a intestinal bacteria (can not express NDM-1, negative control).In the time that No. 1 scraps of paper only add Ampicillin Trihydrate, almost can not suppress the colibacillary growth of NDM-1; In the time that the 2-4 scraps of paper add respectively the compound of Ampicillin Trihydrate and different concns, antibiotic antimicrobial effect has recovered, and can suppress the colibacillary growth of NDM-1; No. 5 scraps of paper add the zine ion that EDTA can complexing NDM-1, thereby have suppressed the hydrolytic activity of NDM-1, make Ampicillin Trihydrate can suppress the colibacillary growth of NDM-1; No. 6 the scraps of paper have added L-Cap positive drug, can suppress NDM-1 activity, make Ampicillin Trihydrate can suppress the colibacillary growth of NDM-1; No. 7 the scraps of paper only add compound, and visible A and B all do not have inhibition zone, illustrate that compound itself does not have bacteriostatic activity.
Claims (7)
1. following formula (I) represents compound or its salt or solvate are used as the purposes in the medicine of NDM-1 metallo-β-lactamase inhibitor in preparation:
Wherein
R
1represent hydrogen atom or C
1-6alkyl, all these groups can be substituted;
R
2represent hydrogen atom, C
1-8alkyl, C
3-8cycloalkyl, aryl, iso-aryl, phenyl-C
1-6alkyl, phenylamino acyl group, C
3-8cycloalkyl-C
1-6alkyl, iso-aryl-C
1-6alkyl, all these groups can be substituted;
R
3represent hydrogen atom, cyano group, nitro, halogen atom, C
1-6alkyl, C
1-6haloalkyl, C
1-6hydroxyalkyl, C
1-6aminoacyl, C
3-7cycloalkyl, aryl, iso-aryl, different cycloalkyl, hydroxyl, carbonyl, ester group, amino, all these groups can be substituted.
2. the pharmaceutical composition that comprises compound that general formula (I) that claim 1 defines represents and pharmaceutically acceptable carrier in preparation as the purposes in the medicine of NDM-1 metallo-β-lactamase inhibitor.
3. the pharmaceutical composition that comprises compound that general formula (I) that claim 1 defines represents and pharmaceutically acceptable carrier together with the microbiotic of Ampicillin Trihydrate in preparation as the purposes in the medicine of beta-lactam antibiotics.
4. the pharmaceutical composition that comprises compound that general formula (I) that claim 1 defines represents and pharmaceutically acceptable carrier together with the microbiotic of Ampicillin Trihydrate in preparation as the purposes in antibiotic medicine, wherein said pharmaceutical composition is with the Ampicillin Trihydrate microbiotic while or use successively.
5. the pharmaceutical composition that comprises compound, Ampicillin Trihydrate microbiotic and optional pharmaceutically acceptable carrier that general formula (I) that claim 1 defines represents in preparation as the purposes in antibiotic medicine.
6. the purposes of the compound that the general formula (I) that Ampicillin Trihydrate microbiotic and claim 1 define represents in the medicine infecting for the preparation for the treatment of bacterium, the compound that the general formula (I) that wherein said Ampicillin Trihydrate microbiotic and claim 1 define represents is used jointly.
7. the compound that claim 1 defines is the purposes in the medicine for the preparation of prevention or the infection for the treatment of bacterium together with the microbiotic of Ampicillin Trihydrate.
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| CN201410166150.XA CN103951680A (en) | 2014-04-24 | 2014-04-24 | Application of novel metal beta-lactamase inhibitor in preparation of medicines for resisting drug-resistance bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110121349A (en) * | 2016-09-28 | 2019-08-13 | 香港大学 | Bismuth (III) compounds and methods |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110121349A (en) * | 2016-09-28 | 2019-08-13 | 香港大学 | Bismuth (III) compounds and methods |
| CN110121349B (en) * | 2016-09-28 | 2022-08-23 | 香港大学 | Bismuth (III) compounds and methods thereof |
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