CN105399680B - Chiral more PCBN tools and preparation method and application - Google Patents
Chiral more PCBN tools and preparation method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
- C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
- C07C233/47—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
Abstract
The invention discloses a kind of chiral more PCBN tools and preparation method and application, belong to protein inhibitor technical field.Solve in the prior art, POMs lacks targeting, and recognition capability is poor under the biotic environment system of complexity, can not realize the technical problem of clinical practice.Chiral more PCBN tools of the present invention, shown in structural formula such as formula (I), in formula, R is L His, D His, L Asp, D Asp, L Leu, D Leu, L Phe or D Phe take off the residue of an amino hydrogen.The more PCBN tools of the chirality can be used as A beta inhibitor applications, can penetrate blood-brain barrier, realize under physiological environment, have high affinity and selectivity to A β, and the means of more horn of plenty are provided for AD treatment.
Description
Technical field
The invention belongs to protein inhibitor technical field, and in particular to a kind of chiral more PCBN tools and its preparation side
Method and application.
Background technology
Alzheimer disease (Alzheimer ' s disease, abbreviation AD) is a kind of progress sexual development of onset concealment
Nervous system degenerative disease.Clinical manifestation is that the acquired Premium Features of cerebral cortex are damaged, i.e., dull-witted, mainly including different journeys
The failure of memory of degree, perceptibility, judgment, thinking ability, locomitivity etc. is impaired, and emotional responses obstacle and personality
Change.At present, AD kills as the fourth-largest of human health that cardiovascular disease, cancer and headstroke are only second in modern society
Hand.Counted according to the World Health Organization, the expense of beginning of this century whole world treatment AD patient accounts for disease population total expenditure in more than 60 years old
11.2%, far above above-mentioned three.By the end of 2013, existing more than 40,000,000 AD patient, wherein China in global range
AD patient be 10,000,000, absolute quantity occupy first place in the world.According to the prediction of epidemiology, to the quantity meeting of the year two thousand fifty patient
Increase to about 100,000,000.With the continuous improvement of people's living standard, many countries including China or will enter
Enter aging society, the ratio of elderly population will increasingly rise, and objectively also increase AD potential patient groups.And with AD
For representative, the incidence of disease of the nerve degenerative diseases in the elderly that increases with the age and occur remain high, it is most
Patient is the old man of over-65s, and the course of disease grows (generally 10-20, or even longer), without effective treatment means, to society
Meeting, family all bring white elephant.
The major pathologic features of Alzheimer's disease are：The position such as temporal lobe and hippocampal cortex neuron loss, mainly by courage
Alkali energy function damage causes；Neurofibrillary tangles (neurofibrillary tangles, NFTs) is mainly related by micro-pipe
Albumen tau Hyperphosphorylationof and glycoforms condenses generation in the cell；Extracellular senile plaque expelling (the senile of intracerebral
Plaques, SPs) deposition, typical senile plaque expelling core assemble many a diameter of 8-10nm or so by amyloid-beta
The fiber that (β-amyloid peptide, abbreviation A β) aggregation forms, the nutritious bad nervous process of surrounding, the star of activation
Spongiocyte and microglia surround the fibrous plaque for forming densification.
AD definite pathogenesis is also clear without studying.There are a variety of theories at present for the AD cause of disease.Wherein, 1992
The A β cascade hypothesis (amyloid cascade hypothesis) that Hardy and Higgins is proposed are of greatest concern, and they think A β
Aggregation and Energy Deposition aggravate neurofibrillar entanglement, and cause cell death, be the main reason that AD is formed.By exempting from
After the patch that A beta peptide aggregations form is removed in epidemic disease injection, the nervous system lesion related to AD can be repaired, and this is that A β cascades hypothesis carries
Strong evidence is supplied.But in organic molecule of the prior art and peptide inhibitor, only seldom a part of peptides
Inhibitor can realize the Selective recognition to A β, and this kind of inhibitor is often difficult to penetrate blood-brain barrier.This is allowed for
Inhibitor is stated to be difficult to obtain the effect of good in clinical treatment.
2011, Angew.Chem.Int.Ed. (German applied chemistry) magazine, disclose a series of poly oxometallic acids
Salt (also known as heteropoly acid, POMs), more PCBN tools of the series are negatively charged in physiological conditions, can be with A β positive electricity area
His13-Lys16 (HHQK) produces an electrostatic interaction.Therefore, more PCBN tools can regulate and control AD paraproteins
A β aggregation.But POMs lacks targeting as inorganic compound, under the biotic environment system of complexity recognition capability compared with
Difference, this allows for them still has a certain distance between actual clinical practice.
The content of the invention
Present invention aim to address in the prior art, POMs lacks targeting, knows under the biotic environment system of complexity
Other ability is poor, can not realize the technical problem of clinical practice, there is provided a kind of chiral more PCBN tools and preparation method thereof.
It is as follows that the present invention solves the technical scheme that above-mentioned technical problem uses.
Chiral more PCBN tools, structural formula are as follows：
In formula ,-R is one kind in following structure：
The preparation method of chiral more PCBN tools, comprises the following steps：
(1) amination Anderson types POM and succinic anhydride are pressed to the amount 1 of material:(2-10) is dissolved in anhydrous acetonitrile,
Reaction more than 24h is stirred at room temperature, removes precipitation, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as carboxylated
Anderson types POM；
(2) by carboxylated Anderson types POM, n-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC)
By the amount 1 of material:1:1 is dissolved in anhydrous DMF (DMF), and reaction more than 24h is stirred at room temperature, and it is heavy to remove
Form sediment, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as activated carboxylic Anderson types POM；
(3) activated carboxylic Anderson types POM and chiral amino acid are pressed to the amount ratio 1 of material:(1-20) is dissolved in anhydrous
In DMF, reaction more than 48h is stirred at room temperature, removes precipitation, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as hand
The more PCBN tools of property；
The chiral amino acid is L-His, D-His, L-Asp, D-Asp, L-Leu, D-Leu, L-Phe or D-Phe.
Preferably, in step (1), mixing speed is more than 200r/min, mixing time 24-48h.
Preferably, in step (2), mixing speed is more than 200r/min, mixing time 24-48h.
Preferably, in step (3), mixing speed is more than 200r/min, mixing time 48-72h.
The above-mentioned more PCBN tools of chirality can be used as amyloid-beta inhibitor.
Compared with prior art, beneficial effects of the present invention：
1st, chiral more PCBN tools of the invention have POM structures and chiral amino acid structure, in physiological conditions,
Negatively charged POM structures and A β positive electricity area His13-Lys16 (HHQK) produce an electrostatic interaction, meanwhile, modified on POM
Small alkane chain and A β 18 two amino acid of Leu 17 and Val produce water deliverys interaction；Chiral amino acid structure and A β
The amino acid of respective regions produce chiral Recognition effect, these three active forces play a role so that such chiral poly jointly
There is a very big binding constant between oxometallate and A β, so as to realize the chiral poly oxometallic acid of the present invention
High sensitivity of the salt to A β, strong specific recognition capability, through experimental tests, modify the Anderson types POM's of chiral amino acid
Binding ability is eager to excel 16-1090 times than unmodified Anderson types POM, and it is very strong two hydrophobicitys in A β sequences to be present
Amino acid Phe19 and Phe20, if the chiral amino acid modified on POM is hydrophobic amino acid, it can further improve knot
Close binding ability；
2nd, chiral more PCBN tools of the invention are good to the inhibition of A beta peptide aggregations, than unmodified Anderson
Type POM is eager to excel 37-226 times；
3rd, chiral more PCBN tools of the invention, can eliminate the beta mediated cytotoxicities of A；
4th, chiral more PCBN tools of the invention can penetrate the suppression of blood-brain barrier as A beta inhibitor applications
Agent, realize under physiological environment, to A β high affinity and selectivity, the means of more horn of plenty are provided for AD treatment.
Brief description of the drawings
The THT curves that the chiral POM that Fig. 1 is 1-8 of the embodiment of the present invention is added after the hatching of A β culture mediums；
Fig. 2 is the influence of 1-8 of the embodiment of the present invention chiral POM cytotoxicities beta mediated to A；
Fig. 3 is effect of vigor of the POM-D-Phe to PC12 cells of various concentrations；
Fig. 4 is MnMo in the chiral more PCBN tools of the present invention6O24Floor map；
In figure, 1-6 all represents key.
Embodiment
The invention thought of the present invention：2011, it is (also known as miscellaneous that German applied chemistry discloses a series of more PCBN tools
Polyacid, POMs), more PCBN tools of the series are negatively charged in physiological conditions, can be with A β positive electricity area His13-
Lys16 (HHQK) produces an electrostatic interaction.2009, PNAS reports small alkane chain can be with A β
18 two amino acid of Leu 17 and Val produce water deliverys interaction.In A β sequences, ensuing two amino acid is
Phe19 and Phe20, this is the very strong amino acid of two hydrophobicitys, can be with if modifying hydrophobic amino acid on POM
They produce interaction, and then obtain more preferable targeting.Simultaneously as the natural amino acid in organism is chiral
L-type amino acid, so for any natural protein, there is chiral microenvironment around it, this is equally applicable to
Aβ.If modifying chiral structure on POM, interacted with A β chiral microenvironment, it is possible to obtain stronger combination energy
Power.Consideration application environment is physiological environment, so paying the utmost attention to chiral amino acid.But different amino acid have it is different
Property, also will not be identical for A β binding ability after modifying on POM, so the selection of amino acid is particularly significant.Even and if
It is same amino acid, different chiralitys also has different binding abilities, and a kind of chiral compound can be used as medicine, still
Its enantiomer is invalid (such as world-shaking Japanese " reaction stops " medical event) that even may be poisonous, so amino
Sour chiral selection is also particularly significant.Contemplate based on more than, we have designed and synthesized following POM derivatives.
Chiral more PCBN tools of the present invention, shown in structural formula such as formula (I)：
In formula ,-R is that L-His, D-His, L-Asp, D-Asp, L-Leu, D-Leu, L-Phe or D-Phe slough one
Residue after amino hydrogen, specific R are one kind in following structure：
Wherein, chemical formula is identical, two kinds of structures of symmetrical configuration mapping structure each other, that is, possesses different chiralitys, i.e., on
State in structure, from left to right, from top to bottom, represent L-His, D-His, L-Asp, D-Asp, L-Leu, D-Leu, L-Phe successively
Or D-Phe sloughs the residue after an amino hydrogen.When-R is the residue after D-Phe sloughs an amino hydrogen, chiral poly
The effect of oxometallate is carried out, and binding ability is more eager to excel in whatever one does by 10 than the Anderson types POM modified without chiral amino acid3Times, institute
Residue after sloughing an amino hydrogen as D-Phe using preferred R.
It should be noted that in chiral more PCBN tools of the present invention, due to MnMo6O24For cage structure, so
It can not be embodied by structure, generally use formula (II) is illustrated to it in the prior art, for details, reference can be made to document A collection
ofrobust methodologies for the preparation of asymmetric hybrid Mn–Anderson
polyoxometalates for multifunctional materials(Chem.Sci.,2013,4,3810–3817.)。
MnMo6O24Structural plan schematic diagram as shown in figure 4, from fig. 4, it can be seen that MnMo6O246 MoO4Impaled by coordinate bond
Carry out a Mn atom, then 3 C of 1,3,5 number keys and the same side form C-O keys respectively；No. 2,4,6 and 3 C points of opposite side
Xing Cheng not C-O keys.
The preparation method of chiral more PCBN tools of the invention, comprises the following steps：
(1) amination Anderson types POM and succinic anhydride are pressed to the amount 1 of material:(2-10) is dissolved in anhydrous acetonitrile,
Reaction more than 24h is stirred at room temperature, general 200-400r/min rotating speeds stir 24-48h, remove precipitation, and filtrate is placed in ether atmosphere
In crystallized, obtain crystal, as carboxylated Anderson types POM；
(2) carboxylated Anderson types POM, NHS and DCC are pressed to the amount 1 of material:1:1 is dissolved in dry DMF, room temperature
More than stirring reaction 24h, general 200-400r/min rotating speeds stir 24-48h, remove precipitation, and filtrate is placed in ether atmosphere
Row crystallization, obtains crystal, as activated carboxylic Anderson types POM；
(3) activated carboxylic Anderson types POM and chiral amino acid are pressed to the amount ratio 1 of material:(1-20) is dissolved in anhydrous
In DMF, reaction more than 48h is stirred at room temperature, general 200-400r/min rotating speeds stir 48-72h, remove precipitation, and filtrate is placed in second
Crystallized in ether atmosphere, obtain crystal, be chiral more PCBN tools；
Wherein, chiral amino acid L-His, D-His, L-Asp, D-Asp, L-Leu, D-Leu, L-Phe or D-Phe,
It is preferred that D-Phe.
It should be noted that amination Anderson types POM is prior art, structural formula is [N (C4H9)4]3[MnMo6O18
{(OCH2)3CNH2}2], it can be prepared by existing method, referring to document A collection of robust
methodologies for the preparation of asymmetric hybrid Mn–Anderson
polyoxometalates for multifunctional materials(Chem.Sci.,2013,4,3810–3817.)。
The present invention provides a kind of amination Anderson types POM preparation method, but not limited to this, comprises the following steps that：
(1) by Na2MoO4·2H2O is dissolved in secondary water, is added excessive hydrochloric acid with vigorous stirring, is then proceeded to
Room temperature is stirred vigorously 10min, obtains solution A；
(2) TBAB is dissolved in secondary water, obtains solution B；
(3) it is stirred vigorously down, solution B is added dropwise in solution A, heating stirring reaction 1h, collected by suction precipitation, uses
Secondary water washing 3 times, what is obtained is precipitated and dissolved in the acetone of boiling, and heat filters out insoluble matter, and filtrate is placed in -20 DEG C of precipitations,
Filter, after 2 ether wash, dry, obtain solid；
(4) solid for obtaining step (3), excessive MnAc3·2H2O and Tris, is dissolved in anhydrous acetonitrile, heats back jointly
Stream, after being cooled to room temperature, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and gained crystal is amination
Anderson types POM.
Chiral more PCBN tools of the invention can be used as A beta inhibitors, and chiral POM is incorporated in A β HHQKLVFF areas
Domain.Binding site of the inhibitor on A β can be determined using identical titration calorimetry (ITC).
The present invention is further illustrated with reference to embodiments.
Embodiment 1
Chiral more PCBN tools, shown in structural formula such as formula (I), in formula (I), R is
The preparation method of above-mentioned inhibitor, comprises the following steps：
(1) amination Anderson types POM and succinic anhydride are pressed to the amount 1 of material:2 are dissolved in anhydrous acetonitrile, room temperature
Stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as carboxylated Anderson types
POM；
(2) carboxylated Anderson types POM, NHS and DCC are pressed to the amount 1 of material:1:1 is dissolved in dry DMF, room temperature
Stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as activated carboxylic Anderson
Type POM；
(3) activated carboxylic Anderson types POM and L-His are pressed to the amount ratio 1 of material:1 is dissolved in dry DMF, room temperature
Stirring reaction 48h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as chiral poly oxometallic acid
Salt, it is designated as POM-L-His.
Embodiment 2
Chiral more PCBN tools, shown in structural formula such as formula (I), R is
The preparation method of above-mentioned inhibitor, comprises the following steps：
(1) amination Anderson types POM and succinic anhydride are pressed to the amount 1 of material:4 are dissolved in anhydrous acetonitrile, room temperature
Stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as carboxylated Anderson types
POM；
(2) carboxylated Anderson types POM, NHS and DCC are pressed to the amount 1 of material:1:1 is dissolved in dry DMF, room temperature
Stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as activated carboxylic Anderson
Type POM；
(3) activated carboxylic Anderson types POM and D-His are pressed to the amount ratio 1 of material:5 are dissolved in dry DMF, room temperature
Stirring reaction 48h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as chiral poly oxometallic acid
Salt, it is designated as POM-D-His.
Embodiment 3
Chiral more PCBN tools, shown in structural formula such as formula (I), R is
The preparation method of above-mentioned inhibitor, comprises the following steps：
(1) amination Anderson types POM and succinic anhydride are pressed to the amount 1 of material:5 are dissolved in anhydrous acetonitrile, room temperature
Stirring reaction 36h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as carboxylated Anderson types
POM；
(2) carboxylated Anderson types POM, NHS and DCC are pressed to the amount 1 of material:1:1 is dissolved in dry DMF, room temperature
Stirring reaction 36h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as activated carboxylic Anderson
Type POM；
(3) activated carboxylic Anderson types POM and D-Asp are pressed to the amount ratio 1 of material:5 are dissolved in dry DMF, room temperature
Stirring reaction 48h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as chiral poly oxometallic acid
Salt, it is designated as POM-D-Asp.
Embodiment 4
Chiral more PCBN tools, shown in structural formula such as formula (I), R is
The preparation method of above-mentioned inhibitor, comprises the following steps：
(1) amination Anderson types POM and succinic anhydride are pressed to the amount 1 of material:6 are dissolved in anhydrous acetonitrile, room temperature
Stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as carboxylated Anderson types
POM；
(2) carboxylated Anderson types POM, NHS and DCC are pressed to the amount 1 of material:1:1 is dissolved in dry DMF, room temperature
Stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as activated carboxylic Anderson
Type POM；
(3) activated carboxylic Anderson types POM and L-Asp are pressed to the amount ratio 1 of material:8 are dissolved in dry DMF, room temperature
Stirring reaction 60h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as chiral poly oxometallic acid
Salt, it is designated as POM-L-Asp.
Embodiment 5
Chiral more PCBN tools, shown in structural formula such as formula (I), R is
The preparation method of above-mentioned inhibitor, comprises the following steps：
(1) amination Anderson types POM and succinic anhydride are pressed to the amount 1 of material:6 are dissolved in anhydrous acetonitrile, room temperature
Stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as carboxylated Anderson types
POM；
(2) carboxylated Anderson types POM, NHS and DCC are pressed to the amount 1 of material:1:1 is dissolved in dry DMF, room temperature
Stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as activated carboxylic Anderson
Type POM；
(3) activated carboxylic Anderson types POM and L-Leu are pressed to the amount ratio 1 of material:20 are dissolved in dry DMF, room temperature
More than stirring reaction 72h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as chiral poly metal
Oxygen hydrochlorate, is designated as POM-L-Leu.
Embodiment 6
Chiral more PCBN tools, shown in structural formula such as formula (I), R is
The preparation method of above-mentioned inhibitor, comprises the following steps：
(1) amination Anderson types POM and succinic anhydride are pressed to the amount 1 of material:10 are dissolved in anhydrous acetonitrile, room temperature
Stirring reaction 48h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as carboxylated Anderson types
POM；
(2) carboxylated Anderson types POM, NHS and DCC are pressed to the amount 1 of material:1:1 is dissolved in dry DMF, room temperature
Stirring reaction 48h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as activated carboxylic Anderson
Type POM；
(3) activated carboxylic Anderson types POM and D-Leu are pressed to the amount ratio 1 of material:10 are dissolved in dry DMF, room temperature
Stirring reaction 48h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as chiral poly oxometallic acid
Salt, it is designated as POM-D-Leu.
Embodiment 7
Chiral more PCBN tools, shown in structural formula such as formula (I), R is
The preparation method of above-mentioned inhibitor, comprises the following steps：
(1) amination Anderson types POM and succinic anhydride are pressed to the amount 1 of material:(2-10) is dissolved in anhydrous acetonitrile,
Reaction more than 24h is stirred at room temperature, removes precipitation, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as carboxylated
Anderson types POM；
(2) carboxylated Anderson types POM, NHS and DCC are pressed to the amount 1 of material:1:1 is dissolved in dry DMF, room temperature
More than stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as activated carboxylic
Anderson types POM；
(3) activated carboxylic Anderson types POM and L-Phe are pressed to the amount ratio 1 of material:(1-20) is dissolved in dry DMF,
Reaction more than 48h is stirred at room temperature, removes precipitation, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as chiral poly
Oxometallate, it is designated as POM-L-Phe.
Embodiment 8
Chiral more PCBN tools, shown in structural formula such as formula (I), R is
The preparation method of above-mentioned inhibitor, comprises the following steps：
(1) amination Anderson types POM and succinic anhydride are pressed to the amount 1 of material:8 are dissolved in anhydrous acetonitrile, room temperature
Stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as carboxylated Anderson types
POM；
(2) carboxylated Anderson types POM, NHS and DCC are pressed to the amount 1 of material:1:1 is dissolved in dry DMF, room temperature
Stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as activated carboxylic Anderson
Type POM；
(3) activated carboxylic Anderson types POM and D-Phe are pressed to the amount ratio 1 of material:15 are dissolved in dry DMF, room temperature
Stirring reaction 60h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as chiral poly oxometallic acid
Salt, it is designated as POM-D-Phe.
The chiral more PCBN tool progressive obtained to embodiment 1-8 can be tested
1st, the binding ability of chiral more PCBN tools and A β that measure embodiment 1-8 is obtained
The Nano ITC instruments produced using TA companies of the U.S. are measured, and are 20 μM of 1.4ml in the sample cell of instrument lower section
A β solution；Derive in over-injected pin for the POM synthesized by 448 μM of 250ml Anderson types POM or embodiment 1-8
Thing, carry out identical titration calorimetry (ITC).As a result show by modifying amino acid of different nature, POM pairs can be strengthened really
In A β binding ability, while even same amino acid, different chiralitys also have different binding abilities.Wherein water delivery ammonia
Lifting of the modification of base acid for binding ability is beneficial, and lifting of the modification of D type amino acid for binding ability is also same
Sample is beneficial.So the POM (POM-D-Phe) of D-Phe modifications possesses most strong binding ability.
2nd, thioflavin T hT is tested
A β kinetics of aggregation can be detected by the change of ThT fluorescence.If A β assemble, then ThT fluorescence
It will rise；If A β aggregation is suppressed, then ThT fluorescence would not occur significantly to change.ThT fluorescence exciting wavelengths
In 444nm, launch wavelength is in 482nm.
With 50mM HEPES (pH 7.4, including 150mM NaCl) for solvent, 40 μM of A β solution are configured, a copy of it is existed
37 DEG C are incubated, remaining the eight parts amination Anderson types POM, POM-D-His for being separately added into 20 μM, POM-L-His,
POM-D-Asp, POM-L-Asp, POM-L-Leu, POM-D-Leu, POM-L-Phe, POM-D-Phe, equally incubated at 37 DEG C
Educate.Sampled every 24h and add ThT, the ThT fluorescence of above-mentioned ten parts of solution is detected using Fluorescence spectrophotometer.Testing conditions
For, final concentration of 1 μM of A β's in colorimetric pool, final concentration of 10 μM of probe molecule ThT, cumulative volume 300ul.As a result such as Fig. 1
It is shown.In Fig. 1, curve is followed successively by A β when individually hatching from top to bottom, add amination Anderson types POM after hatching, add
Hatch after POM-D-His after hatching, addition POM-L-His after hatching, addition POM-D-Asp after hatching, addition POM-L-Asp,
Add POM-L-Leu hatching after, add POM-D-Leu hatching, add POM-L-Phe after hatching, add POM-D-Phe after hatch
ThT fluorescence curves.It will be seen from figure 1 that A β, individually when being incubated for 37 DEG C, the S types that ThT fluorescence shows characteristic are bent
Line.After chiral POM is added, the formation of A beta peptide aggregation bodies is significantly inhibited.It can be seen according to the increased speed of ThT fluorescence
Go out, inhibition it is best be still POM-D-Phe, compared with unmodified POM, 503nhibiting concentrations of the POM-D-Phe to A β
(IC50) it have dropped more than 200 times.
3rd, chiral POM suppresses the performance detection of the beta mediated bio-toxicities of A
PC12 cells are that we analyze chiral POM pairs with mtt assay for studying a kind of more nerve cell of A β toxicity
The influence of A β neurotoxicities.
2 μM of A β, 2 μM of A β and 1 μM of POM-D-Phe, 2 μM of A β and 1 μM of POM-D-Phe are buffered in vitro respectively molten
In liquid 37 DEG C incubation 1h, then take equivalent three kinds of solution be distributed to respectively in PC12 culture medium cultivate 24h (blank sample be by
The buffer solution of equivalent is added in PC12 culture medium), then add the MTT of equivalent into culture medium respectively and continue to cultivate 4h, remove
Purple crystals obtained by after culture medium are dissolved with dimethyl sulfoxide (DMSO) (DMSO), are finally determined with ELIASA under 490nm wavelength
Absorption value, as a result as shown in Figure 2.Figure it is seen that A beta peptide aggregations can cause cell viability reduce about 40%, chiral POM with
A β hatch the toxicity that can significantly reduce A β, and cell viability can at most be lifted when especially incubation altogether with POM-D-Phe
To more than 90%.
Take the PC12 cells that five parts of identicals contain culture medium, be separately added into final concentration of 0 μM, 10 μM, 20 μM, 50 μM,
100 μM of POM-D-Phe is incubated 24h at 37 DEG C, then into culture medium, the MTT of addition equivalent continues to cultivate 4h, removes culture medium
Resulting purple crystals dimethyl sulfoxide (DMSO) (DMSO) dissolves afterwards, determines the absorption value under 490nm wavelength with ELIASA, as a result
As shown in Figure 3.From figure 3, it can be seen that chiral POM is little to the effect of vigor of PC12 cells in itself.
It can illustrate with reference to Fig. 2 and Fig. 3, chiral POM can not only suppress A beta peptide aggregations in vitro, still have in the cell
Effect.
It should be noted that the A β that test experiments of the present invention use all pass through pre-treatment, pretreatment process is first by A β powder
End is dissolved in hexafluoroisopropanol with 1mg/ml concentration, then seals bottleneck, is vibrated 2h at 4 DEG C, is promoted it to be completely dissolved, then
Storing solution is frozen in -20 DEG C of refrigerators.Before the use, hexafluoroisopropanol is dried up with soft nitrogen stream, then again
It is dissolved in corresponding cushioning liquid, balance 6h is stood in 4 DEG C of refrigerators.The PC12 cells that test experiments of the present invention use exist
Cultivated in the culture mediums of RPMI 1640,5%CO is contained in incubator2After cultivating 72h, 1000rpm centrifugations 5min collects cell, uses
PBS cleans twice.Cell is resuspended in the cushioning liquid of pH 7.4, with Petroff-Hausser cell counters pair
Cell is counted.
Claims (6)
1. chiral more PCBN tools, it is characterised in that structural formula is as follows：
In formula ,-R is one kind in following structure：
2. the preparation method of chiral more PCBN tools described in claim 1, it is characterised in that comprise the following steps：
(1) amination Anderson types POM and succinic anhydride are pressed to the amount 1 of material:(2-10) is dissolved in anhydrous acetonitrile, room temperature
More than stirring reaction 24h, precipitation is removed, filtrate is placed in ether atmosphere and crystallized, and obtains crystal, as carboxylated
Anderson types POM；
(2) carboxylated Anderson types POM, n-hydroxysuccinimide and dicyclohexylcarbodiimide are pressed to the amount 1 of material:1:
1 is dissolved in anhydrous DMF, and reaction more than 24h is stirred at room temperature, removes precipitation, filtrate is placed in ether atmosphere
Crystallized, obtain crystal, as activated carboxylic Anderson types POM；
(3) activated carboxylic Anderson types POM and chiral amino acid are pressed to the amount ratio 1 of material:(1-20) is dissolved in anhydrous N, N-
In dimethylformamide, reaction more than 48h is stirred at room temperature, removes precipitation, filtrate is placed in ether atmosphere and crystallized, and obtains crystalline substance
Body, it is chiral more PCBN tools；
The chiral amino acid is L-His, D-His, L-Asp, D-Asp, L-Leu or D-Leu.
3. the preparation method of the more PCBN tools of chirality according to claim 2, it is characterised in that in step (1), stir
It is more than 200r/min, mixing time 24-48h to mix speed.
4. the preparation method of the more PCBN tools of chirality according to claim 2, it is characterised in that in step (2), stir
It is more than 200r/min, mixing time 24-48h to mix speed.
5. the preparation method of the more PCBN tools of chirality according to claim 2, it is characterised in that in step (3), stir
It is more than 200r/min, mixing time 48-72h to mix speed.
6. application of the chiral more PCBN tools in amyloid-beta inhibitor is prepared described in claim 1.
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| CN111072722B (en) * | 2019-12-27 | 2022-08-16 | 湖北工业大学 | Anderson polyacid and application thereof as CVB3 virus inhibitor |
| CN111138499B (en) * | 2019-12-27 | 2022-10-04 | 湖北工业大学 | Anderson polyacid and application thereof in resisting ADV7 virus |
| CN111072723B (en) * | 2019-12-27 | 2022-10-11 | 湖北工业大学 | Anderson polyacid organic derivative modified by monoiodo benzoic acid through covalent bond and application of Anderson polyacid organic derivative in resisting ADV7 virus |
| CN113600240B (en) * | 2021-07-16 | 2022-08-19 | 清华大学 | Amino acid or derivative modified polyoxometallate and preparation method and application thereof |
| CN115300527B (en) * | 2022-03-30 | 2023-09-26 | 河北医科大学 | Application of chiral heteropolyacid enantiomer-selective inhibition of beta-amyloid peptide aggregation in preparation of drug for treating Alzheimer's disease |
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