CN101785773B - Use of metal supermolecular compound in preparation of medicament for treating alzheimer's disease - Google Patents
Use of metal supermolecular compound in preparation of medicament for treating alzheimer's disease Download PDFInfo
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Abstract
The invention relates to the technical field of supermolecular compounds and discloses the use of the metal supermolecular compounds in the preparation of the medicament for treating alzheimer's disease. The core metal ions of the metal supermolecular compound are all Ni<2+>s or Fe<2+>s; the ligands of the metal supermolecular compound have a molecular formula of C25H20N4. In a mole of the metal supermolecular compound, two metal positive ions and three imine-based ligands are bonded by coordinate bonds; and the Fe<2+>s and the N form a stable hexa-coordinate structure or the Ni<2+>s and the N form a stable hexa-coordinate structure so as to form a double-core metal three-spiral cylinder structure. The central phenylene surfaces of the metal supermolecular compound interact with the aromatic branch chains of the A beta amino acid residues through hydrophobic effect and the action of Pi bonds. The charge parts at two ends of each mole of the metal supermolecular compound can be bonded with the A beta amino acid residues by electrostatic interaction so as to inhibit the aggregation of the A beta amino acid residues.
Description
Technical field
The present invention relates to the super molecular compound technical field, more particularly, relate to the application of metal supermolecular compound in the medicine of preparation treatment Alzheimer.
Background technology
Alzheimer (being called for short AD) is a kind of constitutional central nervous system degenerative disease that betides human geratic period and presenium.The Clinical symptoms of Alzheimer's disease is that the acquired Premium Features of cerebral cortex are impaired, and is promptly dull-witted.Its main pathology is characterized as: position neuron losses such as temporal lobe and hippocampal cortex, cholinergic function damage; The senile plaque deposition that the brain inner cell is outer; Neurofibrillary tangles; Cerebrovascular amyloid pathological changes.It is amyloid beta (amyloid β-peptide about 8～10nm that typical senile plaque core is being assembled many diameters, be called for short A β) fiber, nutritious on every side bad nervous process, activated astrocyte and microglia are around forming fine and close fibrous plaque.
Discover that accumulative amyloid beta produces neurotoxicity in cell culture medium and in the rat brain, poisons neuron, and harmful to memory.Each links such as the generation of A β, gathering and removing all may become the potential target spot of treatment AD.A β shears the polypeptide with beta sheet configuration that a kind of 39～43 aminoacid of generation are formed by beta-amyloyd precursor protein (APP) in proper order through beta-secretase and gamma-secretase.It is its major reason that causes AD that A β forms polymer by protein monomer by intermolecular gathering.The toxicity of A β is directly related with its coherent condition.
At present, be that the chemical compound of targeting exploitation mostly is the organic molecule material with the A beta peptide aggregation in the prior art, curcumin for example, rifampicin and Congo red etc., the nervous system disease aspect does not reach good effect but it is used for the treatment of Alzheimer etc.An inexpensive approach to supramolecular architecture. (Hannon, M.J. wait Chem.Commun., 1997,18:1807-1808.) in a kind of metal supermolecular compound is disclosed, be for two nuclear metals and three parts by coordinate bond in conjunction with formation, have double-core metal triple helical cylindrical structure, but also do not find its application in preparation treatment Alzheimer medicine at present.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide the application of metal supermolecular compound in preparation treatment Alzheimer medicine.
The invention provides the application of a kind of ferrous metal super molecular compound in the medicine of preparation treatment Alzheimer, described ferrous metal super molecular compound is by two Fe
2+With the part shown in three structural formula as I by coordinate bond in conjunction with the double-core metal triple helical cylindrical structure that forms hexa-coordinate; It is that the Electrospray Mass Spectrometry analysis of mobile phase is presented at mass-charge ratio 310.4 places quasi-molecular ions is arranged with the acetonitrile,
The present invention also provides the application of a kind of nickel metal supermolecular compound in the medicine of preparation treatment Alzheimer, and described nickel metal supermolecular compound is by two Ni
2+With the part shown in three structural formula as I by coordinate bond in conjunction with the double-core metal triple helical cylindrical structure that forms hexa-coordinate; It is that the Electrospray Mass Spectrometry analysis of mobile phase is presented at mass-charge ratio 311.7 places quasi-molecular ions is arranged with the acetonitrile,
From technique scheme as can be seen, metal supermolecular compound nuclear metal provided by the invention is Ni
2+Or be Fe
2+, the ligand molecular formula is C
25H
20N
4, the application in preparation treatment Alzheimer.Described metal supermolecular compound be by two metal cations and three based on the part of imines by coordinate bond combination, Fe
2+Form stable hexa-coordinate structure or Ni with N
2+Form stable hexa-coordinate structure with N, thereby form double-core metal triple helical cylindrical structure.The center phenylene face of metal supermolecular compound and the aromatic side chain of A beta amino acids residue are by hydrophobic or π key effect, and the charged part at its two ends can strengthen and the combining of A β by electrostatic interaction, its bigger space molecular structure can provide steric hindrance for assembling between the A beta molecule in addition, thereby suppresses the gathering of A β.
Sedimentation detects metal supermolecular compound provided by the invention in the accumulative inhibiting experiment of amyloid-beta fibrosis, experimental result shows, A β is hatched a large amount of formation of deposits A β 40 aggregations was taken place in 7 days, and the A β that contains metal supermolecular compound of the present invention under identical incubation conditions forms A β 40 aggregation content and obviously reduces.Thioflavin T (ThT) detects metal supermolecular compound to the accumulative inhibitory action of amyloid-beta fibrosis as fluorescent probe.Experimental result shows that the adding of nickel metal supermolecular compound or ferrous metal super molecular compound can obviously stop the formation of A beta aggregation, and this inhibition effect is relevant with the concentration that adds metal supermolecular compound.Thioflavin T (ThT) detect as fluorescent probe metal supermolecular compound to the accumulative concentration dependency of amyloid-beta fibrosis empirical curve in, calculate the accumulative EC50 value of nickel metal supermolecular compound and ferrous metal super molecular compound inhibition A betaization and be respectively 6.45 μ mol/l and 1.12 μ mol/l, show that metal supermolecular compound provided by the invention is a very promising A beta peptide aggregation inhibitor.Therefore, metal supermolecular compound provided by the invention has good effect aspect the gathering that suppresses A β.Metal supermolecular compound provided by the invention reaches the application aims in the medicine of preparation treatment Alzheimer.
Description of drawings
In order to be illustrated more clearly in the embodiment of the invention or technical scheme of the prior art, to do to introduce simply to the accompanying drawing of required use in embodiment or the description of the Prior Art below, apparently, accompanying drawing in describing below only is some embodiments of the present invention, for those of ordinary skills, under the prerequisite of not paying creative work, can also obtain other accompanying drawing according to these accompanying drawings.
Fig. 1 is the disclosed metal supermolecular compound structural formula of the embodiment of the invention;
Fig. 2 is that the disclosed thioflavin T of the embodiment of the invention (ThT) detects metal supermolecular compound to the accumulative kinetic effect curve of amyloid-beta fibrosis as fluorescent probe, 50 μ mol/l A β, 40 (■), 50 μ mol/ l A β 40 and 1 μ mol/l nickel metal supermolecular compound (●), 50 μ mol/ l A β 40 and 1 μ mol/l ferrous metal super molecular compound (▲);
Fig. 3 is that the disclosed thioflavin T of the embodiment of the invention (ThT) detects metal supermolecular compound to the accumulative concentration dependency of amyloid-beta fibrosis empirical curve as fluorescent probe, nickel metal supermolecular compound (■), ferrous metal super molecular compound (●);
The circular dichroism curve of spectrum that A β 40 secondary structures that Fig. 4 was hatched for the embodiment of the invention is disclosed 1 hour transform with incubation time, A β 40 (■), A β 40 and equimolar nickel metal supermolecular compound (●) and A β 40 and equimolar ferrous metal super molecular compound (▲);
The circular dichroism curve of spectrum that A β 40 secondary structures that Fig. 5 was hatched for the embodiment of the invention is disclosed 4 days transform with incubation time, A β 40 (■), A β 40 and equimolar nickel metal supermolecular compound (●) and A β 40 and equimolar ferrous metal super molecular compound (▲);
The circular dichroism curve of spectrum that A β 40 secondary structures that Fig. 6 was hatched for the embodiment of the invention is disclosed 10 days transform with incubation time, A β 40 (■), A β 40 and equimolar nickel metal supermolecular compound (●) and A β 40 and equimolar ferrous metal super molecular compound (▲);
Fig. 7 causes the Cytotoxic protective effect curve of PC12 for the disclosed metal supermolecular compound of the embodiment of the invention to A β 40;
Fig. 8 is the fluorescence titration curve of disclosed metal supermolecular compound of the embodiment of the invention and A β 40 effects;
Fig. 9 is the depolymerisation curve of the disclosed metal supermolecular compound of the embodiment of the invention to A β 40 fibers that formed, nickel metal supermolecular compound (■), ferrous metal super molecular compound (●);
Figure 10 is hatched 5 hours metal supermolecular compound to the influence curve of established amyloid-beta fiber secondary structure for the disclosed circular dichroism spectral detection of the embodiment of the invention, 50 μ mol/l fA β, 40 (---), 50 μ mol/l fA β 40 add 50 μ mol/l[Ni
2L
3]
4+(---), 50 μ mol/l[Fe
2L
3]
4+(----);
Figure 11 is the influence curve of the disclosed circular dichroism spectral detection of embodiment of the invention metal supermolecular compound to established amyloid-beta fiber secondary structure, [Ni
2L
3]
4+(■) with [Fe
2L
3]
4+(●).
The specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the invention, the technical scheme in the embodiment of the invention is clearly and completely described, obviously, described embodiment only is the present invention's part embodiment, rather than whole embodiment.Based on the embodiment among the present invention, those of ordinary skills belong to the scope of protection of the invention not making the every other embodiment that is obtained under the creative work prerequisite.
The invention provides the application of a kind of ferrous metal super molecular compound in the medicine of preparation treatment Alzheimer, described ferrous metal super molecular compound is by two Fe
2+With the part shown in three structural formula as I by coordinate bond in conjunction with the double-core metal triple helical cylindrical structure that forms hexa-coordinate; It is that the Electrospray Mass Spectrometry analysis of mobile phase is presented at mass-charge ratio 310.4 places quasi-molecular ions is arranged with the acetonitrile.
The present invention also provides the application of a kind of nickel metal supermolecular compound in the medicine of preparation treatment Alzheimer, and described nickel metal supermolecular compound is by two Ni
2+With the part shown in three structural formula as I by coordinate bond in conjunction with the double-core metal triple helical cylindrical structure that forms hexa-coordinate; It is that the Electrospray Mass Spectrometry analysis of mobile phase is presented at mass-charge ratio 311.7 places quasi-molecular ions is arranged with the acetonitrile.
Metal supermolecular compound provided by the invention has structure as shown in Figure 1, and the ligand molecular formula is C
25H
20N
4
A β is an amphipathic peptide.Hydrophobic and electrostatic interaction is contributed very big to gathering between the A beta molecule.Metal supermolecular compound provided by the invention respectively has the positive charge of two units at two ends, three organic ligands are in the hydrophobic inflexible π planar structure of chemical compound intermediate formation.These construction featuress make its can with amphipathic A β peptide effect.F19 is two the important amino acid residues relevant with the A beta peptide aggregation with F20.Be deposited in together by face-Bian π effect at intermediary six the phenylene aromatic rings of metal supermolecular compound structure, provide a strong π face at the chemical compound middle part.This makes metallic compound and A β side chain amino acid residue that strong hydrophobic or π key effect take place easily.Metal supermolecular compound more has superiority than organic molecule: similar micromolecular rigid structure makes this combination and inhibitory action have conformation dependent and big molecular dimension makes chemical compound can stride across a plurality of amino acid residues and adopts multiple combination.In addition, super molecular compound strides across blood brain barrier easily and stability is very high.Therefore, metal supermolecular compound provided by the invention suppresses the gathering of A β, thereby reaches the application aims in the medicine of preparation treatment Alzheimer.
The synthetic method of metal supermolecular compound provided by the invention, can reference literature An inexpensive approach to supramolecular architecture. (Hannon, M.J. wait Chem.Commun., 1997,18:1807-1808.) the method preparation in.
The present invention finds to suppress the accumulative metal supermolecular compound of amyloid-beta fibrosis, comprise that metallic nickel metal supermolecular compound and ferrous metal super molecular compound all can suppress the amyloid-beta fibrosis and assemble, wherein ferrous metal super molecular compound inhibition effect is remarkable.In specific embodiment, the EC50 of nickel metal supermolecular compound, be that the concentration value of amyloid fiber 50% when being suppressed is 6.45 μ mol/l, the EC50 of ferrous metal super molecular compound, the concentration value when promptly amyloid fiber 50% is suppressed are 1.12 μ mol/l.Therefore, the invention provides metal supermolecular compound as the accumulative inhibitor of amyloid-beta fibrosis.And the amyloid-beta fibrosis can produce neurotoxic effect.Metal supermolecular compound suppresses the cytotoxic effect by the generation of amyloid-beta fibrosis accumulation process.In addition, the present invention finds to suppress the amyloid-beta fiber that the accumulative nickel metal supermolecular compound of amyloid-beta fibrosis and ferrous metal super molecular compound can both depolymerization have formed, the wherein better effects if of ferrous metal super molecular compound.In addition, metal supermolecular compound provided by the invention slows down the neurocyte toxicity of amyloid-beta.Therefore, metal supermolecular compound provided by the invention can be used in the application in the medicine of preparation treatment Alzheimer.
In order further to understand technical scheme of the present invention, below in conjunction with embodiment the preferred embodiment of the invention is described, but should be appreciated that these describe just to further specifying the features and advantages of the present invention, rather than to the restriction of claim of the present invention.
The sample preparation of the amyloid-beta of 40 amino acid residues (being called for short A β 40):
Amyloid-beta in the embodiment of the invention is available from Sigma company, and sequence length is 40 amino acid residues.
A β 40 powder dissolutions are 1,1,1,3,3,3, and in-the hexafluoroisopropanol, concentration is 1mg/ml.After the solution sealing,, be stored in-20 ℃ as storing solution 4 ℃ of concussions 2 hours.Before using, after solvent dried up with nitrogen, powder was dissolved in the corresponding buffer solution again, leaves standstill balance 6 hours in 4 ℃ of refrigerators.
The preparation of metal supermolecular compound
The synthetic method reference literature of metal supermolecular compound provided by the invention: An inexpensive approach to supramolecular architecture. (Hannon, Chem.Commun. such as M.J., 1997,18:1807-1808.).Building-up process is as follows:
With 2-pyridine carboxaldehyde and 4,4 '-methylene dianiline (MDA) in alcohol solvent according to 2: 1 mixed in molar ratio, room temperature reaction after 2 hours sucking filtration obtain light yellow product, vacuum drying gets part;
The gained part is mixed according to mol ratio respectively with Nickel dichloride. or iron chloride at 3: 2, reflux is 6 hours in methanol, sucking filtration behind the ammonium hexafluorophosphate of adding equivalent, vacuum drying obtains metal supermolecular compound (nickel metal supermolecular compound productive rate is 75%, and ferrous metal super molecular compound productive rate is 78%).
[Ni
2L
3](PF
6)
4.ESI-MS(CH
3CN)：m/z?311.7([Ni
2L
3]
4+)；Elemental?Anal.：Calcd％：C，49.27；H，3.29；N，9.20；Found％：C，49.48；H，3.32，N，9.17.[Fe
2L
3](PF
6)
4.ESI-MS(CH
3CN)：m/z?310.4([Fe
2L
3]
4+)，420.1([Fe
2L
3(F)]
3+)，1675.5([Fe
2L
3(PF
6)
3]
+)；Elemental?Anal.：Calcd％：C，49.0；H，3.6，N，8.9；Found?C，49.0；H，3.1；N，8.7；
1HNMR(600MHz，CD
3CN，25℃)：δ＝8.96(2H，s，Hi)，8.61(2H，d，H
3)，8.44(2H，t，H
4)，7.81(2H，t，H
5)，7.39(2H，d，H
6)，7.00(4H，br?s，H
ph)，5.59(4H，br?s，H
ph)，4.08(2H，s，CH
2?spacer).
Promptly the nickel metal supermolecular compound is analyzed, is obtained:
Electrospray Mass Spectrometry (acetonitrile): the peak of mass=311.7 is [Ni
2L
3]
4+The peak.
Elementary analysis: the shared percentage ratio of each element, value of calculation are C, 49.27; H, 3.29; N, 9.20; Measured value is C, 49.48; H, 3.32, N, 9.17.Value of calculation is consistent with actual measured value.
The ferrous metal super molecular compound is analyzed, is obtained:
Electrospray Mass Spectrometry (acetonitrile): the peak of mass=310.4 is [Fe
2L
3]
4+The peak; 420.1 be [Fe
2L
3(F)]
3+The peak, 1675.5 is [Fe
2L
3(PF
6)
3]
+The peak.
Wherein, in the mass spectrometric measurement process, relate to the gasification of molecule,, form different fragments, therefore can produce several mass spectra peaks because the stability of molecule is different.[Fe
2L
3(F)]
3+[Fe
2L
3(PF
6)
3]
+In gasification, form.
Elementary analysis: the shared percentage ratio of each element, value of calculation are C, 49.0; H, 3.6, N, 8.9; Measured value is C, 49.0; H, 3.1; N, 8.7.Value of calculation is consistent with actual measured value.
One dimension hydrogen spectrum nuclear-magnetism (600MHz, deuterium is a solvent for acetonitrile, 25 ℃), the one dimension hydrogen spectrum nuclear magnetic data of part as shown in table 1:
The one dimension hydrogen of metal supermolecular compound part spectrum nuclear magnetic data among table 1 embodiment 2
| Chemical shift | The hydrogen number | Peak shape |
| 8.96 | 2 | Singlet |
| 8.61 | 2 | Doublet |
| 8.44 | 2 | Triplet |
| 7.81 | 2 | Triplet |
| 7.39 | 2 | Doublet |
| 7.00 | 4 | Wide unimodal |
| 5.59 | 4 | Wide unimodal |
| 4.08 | 2 | Singlet |
By one dimension hydrogen spectrum nuclear magnetic data analysis in the his-and-hers watches 1, can prove that the part for preparing in the present embodiment is the part shown in I.
Sedimentation detects the metal supermolecular compound of embodiment 2 preparations to the accumulative inhibitory action of amyloid-beta fibrosis, and method is as follows:
The A β 40 of 50 μ mol/l embodiment 1 preparation with etc. the metal supermolecular compound of embodiment 2 preparations of mol ratio hatched aging 7 days at 37 ℃, sample was by (13500rpm) centrifugalize at a high speed 20 minutes.After the centrifugalize, precipitation is resuspended and boil degeneration with buffer solution.Sample dyes colour developing through 12% Tris-tricine sds gel electrophoresis electrophoresis 1 hour under 100V voltage by silver.
Experimental result shows that A β 40 deposits in a large number through aging 7 days generation.A β 40 aggregations mainly exist with monomeric form under SDS degeneration deposition condition, also have the dimer of a small amount of anti-SDS degeneration to occur.The aggregation content that contains the sample of metal supermolecular compound in identical ageing process obviously reduces, the sample that contains the ferrous metal super molecular compound than the aggregation content of the sample that contains the nickel metal supermolecular compound reduce more, show that the metal supermolecular compound of embodiment 2 preparations has well to the accumulative inhibitory action of amyloid-beta fibrosis.
Thioflavin T (ThT) detects metal supermolecular compound to the accumulative inhibitory action of amyloid-beta fibrosis as fluorescent probe.Method is as follows:
A β 40 that 50 μ mol/l embodiment 1 prepare and the sample that wherein contains the metal supermolecular compound (concentration is followed successively by 50 μ mol/l, 10 μ mol/l, 1 μ mol/l, 0.1 μ mol/l, 0.01 μ mol/l) of embodiment 2 preparations are hatched aging at 37 ℃.At aging 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, the ThT fluorescence intensity of 7 days difference working samples.
Fig. 2 is that the disclosed thioflavin T of the embodiment of the invention (ThT) detects metal supermolecular compound to the accumulative kinetic effect curve of amyloid-beta fibrosis as fluorescent probe.As shown in Figure 2, the incubation time that the ThT fluorescence of A β 40 samples strengthens curve and sample is typical S type, shows that it meets coring-dependency Aggregation Model.The adding of nickel metal supermolecular compound or ferrous metal super molecular compound can obviously stop the formation of A β 40 fibrosis aggregations, and this inhibition effect is relevant with the concentration that adds super molecular compound.
Fig. 3 is that the disclosed thioflavin T of the embodiment of the invention (ThT) detects metal supermolecular compound to the accumulative concentration dependency of amyloid-beta fibrosis empirical curve as fluorescent probe.By the ThT fluorescence experiments under the different compound concentrations, as shown in Figure 3, calculate the accumulative EC50 value of nickel metal supermolecular compound and ferrous metal super molecular compound inhibition A β 40 fibrosiss and be respectively 6.45 μ mol/l and 1.12 μ mol/l, show that metal supermolecular compound provided by the invention is very promising A β 40 agglutination inhibitors.
Atomic force microscope detects metal supermolecular compound to the accumulative inhibitory action of amyloid-beta fibrosis.Method is as follows:
The A β 40 of preparation among the embodiment 1 was hatched under 37 ℃ 7 days; With among the embodiment 1 preparation A β 40 and embodiment 2 in the preparation the ferrous metal super molecular compound under 37 ℃, hatched 7 days; With among the embodiment 1 preparation A β 40 and embodiment 2 in the preparation the nickel metal supermolecular compound under 37 ℃, hatched 7 days; Get respectively above-mentioned each 10 μ L of mixture solution of hatching 7 days A β 40 and A β 40 and metal supermolecular compound are added drop-wise on the fresh mica surface of peeling off static 10 minutes, residual liquid is blotted with the edge of a little filter paper bar along drop.Clean sample surfaces 3 times with 50 μ L water immediately then, after this globule of remnants is inhaled and gone and drying in exsiccator with the filter paper bar.Adopt the pattern (Tapping mode) of rapping during the sample image acquisition.
The mixture of mixture, ferrous metal super molecular compound and A β 40 that does not add A β 40 samples, nickel metal supermolecular compound and the A β 40 of chemical compound is carried out atomic force microscope observation respectively, obtain: A β 40 is hatched the back and is formed typical unbranched linear fibre shape structure under 37 ℃, and its length surpasses 1 μ m.The sample that mole nickel super molecular compound such as contains obtains little precipitation and filamentary structure that some are short.When the ferrous metal super molecular compound acts on A β 40, experience hatching of identical time, do not observe aggregate structure substantially.
The inhibitory action that metal supermolecular compound transforms the amyloid-beta secondary structure.Method is as follows:
The A β 40 of preparation among the embodiment 1 is hatched at 37 ℃;
The mixture of metal supermolecular compound of preparation among the A β 40 of preparation among the embodiment 1 and the embodiment 2 is hatched at 37 ℃;
With corresponding buffer solution is that blank is done baseline, and the A β 40 and the A β 40 that get respectively after above-mentioned the hatching measure its circular dichroism spectrum with the blend sample of metal supermolecular compound on JASCO J-810 type circular dichroism spectrogrph.Fig. 4, Fig. 5 and Fig. 6 are hatched the circular dichroism curve of spectrum of A β 40 secondary structures of different time with the incubation time conversion for the embodiment of the invention is disclosed.A β 40, A β 40 and equimolar nickel metal supermolecular compound and A β 40 are hatched different time with the mixture of equimolar ferrous metal super molecular compound under 37 ℃, measure circular dichroism spectrum.
As Fig. 4, Fig. 5 and shown in Figure 6, circular dichroism spectrum shows that nickel metal supermolecular compound and ferrous metal super molecular compound all do not change the coiled structure of A β 40 in conjunction with A β 40.Hatch agingly through 37 ℃, A β 40 slowly transforms to the beta sheet structure.And add nickel metal supermolecular compound or ferrous metal super molecular compound, and the structure of A β 40 still maintains the random coil structure, and two kinds of chemical compounds can both stop the formation of A β 40 beta sheet structures.
Metal supermolecular compound is to the Cytotoxic inhibitory action of amyloid-beta.Method is as follows:
The A β 40 of embodiment 1 preparation was hatched 7 days under 37 ℃ of temperature in external buffer solution according to the sample that different mixed obtains with the nickel metal supermolecular compound of embodiment 2 preparations, the A β 40 and the ferrous metal super molecular compound of embodiment 2 preparations of embodiment 1 preparation.Resulting sample dispersion was cultivated 36 hours in the culture medium of PC12 cell, and the adding final concentration is that the MTT of 0.5mg/mL continues to cultivate 4 hours.Dissolve with DMSO after removing culture medium, measure the absorption value under the 490nm wavelength.
Fig. 7 causes the Cytotoxic protective effect curve of PC12 for the disclosed metal supermolecular compound of the embodiment of the invention to A β 40.As shown in Figure 7, hatch 7 days aging A β 40 samples that obtain through 37 ℃ and can cause 53% PC12 cell death.Nickel metal supermolecular compound and ferrous metal super molecular compound be with A β 40 hatching, the toxicity that can significantly slow down A β, and toxic reduction and metal supermolecular compound have concentration dependent.
Fluorescence titrimetric method is calculated the bond strength of two kinds of metal supermolecular compounds and A β 40 effects
Metal supermolecular compound can cancellation A β 40 tyrosine fluorescence.Fixedly A β 40 concentration are 3 μ mol/l, gradually to wherein dripping chemical compound.Fig. 8 is the fluorescence titration curve of disclosed metal supermolecular compound of the embodiment of the invention and A β 40 effects.As shown in Figure 8, to the mapping of metal supermolecular compound concentration, obtain binding constant with the tyrosine fluorescence signal by nonlinear fitting.
By the method for fluorescence titration, we can be in the hope of bond strength and the metering ratio of chemical compound with A β 40 effects.For nickel metal supermolecular compound and ferrous metal super molecular compound, be all to arrive titration end-point at 1: 1 o'clock in mol ratio, what two kinds of chemical compounds were described is 1: 1 in conjunction with metering than all.The nonlinear fitting titration curve obtains binding constant and is respectively 1.9 * 106M
-1(nickel compound) and 4.9 * 106M
-1(iron compound).
Embodiment 9
Metal supermolecular compound is to the depolymerisation of amyloid-beta fiber.Method is as follows:
The A β 40 of 50 μ mol/l embodiment, 1 preparation is hatched at 37 ℃ and was obtained a fiber in aging 7 days.With a fiber and wherein contain metal supermolecular compound (concentration is followed successively by 50 μ mol/l, 10 μ mol/l, 1 μ mol/l, 0.1 μ mol/l, 0.01 protein sample μ mol/l) is at 37 ℃ of reactions 6 hours, the ThT fluorescence intensity of working sample or circular dichroism spectrum.
Fig. 9 is the depolymerisation curve of the disclosed metal supermolecular compound of the embodiment of the invention to A β 40 fibers that formed, utilizes ThT fluorescence to strengthen experiment and detects the depolymerisation to A β 40 fibers that formed of nickel metal supermolecular compound and ferrous metal super molecular compound.ThT fluorescence enhancing experiment shows that the adding of metal supermolecular compound has obviously reduced the fiber content in the A β sample, and the EC50 value that calculates nickel metal supermolecular compound and ferrous metal super molecular compound depolymerization A β 40 fibers is respectively 7.08 μ mol/l and 1.51 μ mol/l.
Figure 10 is that 50 μ mol/l fA β, 40,50 μ mol/l fA β 40 add 50 μ mol/l[Ni
2L
3]
4+, 50 μ mol/l[Fe
2L
3]
4+37 ℃ of circular dichroism spectrum of hatching after 5 hours; Figure 11 is variable concentrations [Ni
2L
3]
4+[Fe
2L
3]
4+Influence curve to beta sheet structural content among the 50 μ mol/l fA β 40.The circular dichroism spectrum experiment discloses the beta sheet structural content that affiliation reduces A β sample that adds of chemical compound.The A beta monomers that this metal supermolecular compound that illustrates that we use not only can suppress free state is gathered into fiber, also a corpus fibrosum that formed of depolymerization to a certain extent.
From the foregoing description as can be seen, metal supermolecular compound provided by the invention can suppress the amyloid-beta fibrosis and assemble, and wherein ferrous metal super molecular compound inhibition effect is remarkable.And the amyloid-beta fibrosis can produce neurotoxic effect.Metal supermolecular compound suppresses the cytotoxic effect by the generation of amyloid-beta fibrosis accumulation process.In addition, the present invention finds to suppress the amyloid-beta fiber that the accumulative nickel metal supermolecular compound of amyloid-beta fibrosis and ferrous metal super molecular compound can both depolymerization have formed, the wherein better effects if of ferrum super molecular compound.In addition, metal supermolecular compound provided by the invention slows down the neurocyte toxicity of amyloid-beta.Therefore, metal supermolecular compound provided by the invention can be used in the medicine of preparation treatment Alzheimer.
To the above-mentioned explanation of the disclosed embodiments, make this area professional and technical personnel can realize or use the present invention.Multiple modification to these embodiment will be conspicuous concerning those skilled in the art, and defined herein General Principle can realize under the situation that does not break away from the spirit or scope of the present invention in other embodiments.Therefore, the present invention will can not be restricted to these embodiment shown in this article, but will meet and principle disclosed herein and features of novelty the wideest corresponding to scope.
Claims (2)
1. the application of ferrous metal super molecular compound in the medicine of preparation treatment Alzheimer, described ferrous metal super molecular compound is by two Fe
2+With the part shown in three structural formula as I by coordinate bond in conjunction with the double-core metal triple helical cylindrical structure that forms hexa-coordinate; It is that the Electrospray Mass Spectrometry analysis of mobile phase is presented at mass-charge ratio 310.4 places quasi-molecular ions is arranged with the acetonitrile,
2. the application of nickel metal supermolecular compound in the medicine of preparation treatment Alzheimer, described nickel metal supermolecular compound is by two Ni
2+With the part shown in three structural formula as I by coordinate bond in conjunction with the double-core metal triple helical cylindrical structure that forms hexa-coordinate; It is that the Electrospray Mass Spectrometry analysis of mobile phase is presented at mass-charge ratio 311.7 places quasi-molecular ions is arranged with the acetonitrile,
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101328250A CN101785773B (en) | 2010-03-26 | 2010-03-26 | Use of metal supermolecular compound in preparation of medicament for treating alzheimer's disease |
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|---|---|---|---|
| CN2010101328250A CN101785773B (en) | 2010-03-26 | 2010-03-26 | Use of metal supermolecular compound in preparation of medicament for treating alzheimer's disease |
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| Publication Number | Publication Date |
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| Michael J. Hannon et al.An inexpensive approach to supramolecular architecture.《Chemical Communications》.1997,(第18期),第1709-1710页. * |
| 吕亚非.金属-超分子聚合物的合成 结构与应用.《功能高分子学报》.2004 |
| 吕亚非.金属-超分子聚合物的合成,结构与应用.《功能高分子学报》.2004,第17卷(第02期),第307-316页. * |
| 杨银龙等.含吡啶基金属-超分子聚合物研究进展.《高分子通报》.2008,(第01期),第47-53页. * |
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