CN104211947A - Non-linear polymer, and preparation and application thereof - Google Patents
Non-linear polymer, and preparation and application thereof Download PDFInfo
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- CN104211947A CN104211947A CN201310212661.6A CN201310212661A CN104211947A CN 104211947 A CN104211947 A CN 104211947A CN 201310212661 A CN201310212661 A CN 201310212661A CN 104211947 A CN104211947 A CN 104211947A
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Abstract
The invention discloses a non-linear polymer, and a preparation method of the non-linear compound and application thereof. The non-linear new polymerized compound is constructed by utilizing a compound with a branch structure, and by combining the new structure non-covalent bond and correlated protein possessing potential treatment effect and to be subjected to in-vivo screening, protein with potential treatment activity is rapidly conveniently screened in vivo by combining an animal model. When being used for screening protein, the new non-linear compound containing a metal ion structure comprises the following characteristics that 1, screening is rapid and convenient, the time for combining protein for in-vivo activity screening is less than 15 min; and 2, the in-vivo screening process does not change the protein structure, and the new non-linear compound does not have a covalent linkage reaction with protein when screening is performed.
Description
Technical field
The invention discloses preparation method and the purposes of a kind of new non-linear polymer and this polymkeric substance.
Background technology
Protein drug is following very important clinical application treatment means, but at present due to the stability of protein drug, the transformation period is short waits shortcomings, make its determine in vitro potential effectively after effectively cannot screen activity in vivo.That a lot of of great value protein drug cannot be verified in vivo.For screening in body or treatment after having scholar to have the covalently bound albumen of the compound of provide protection with some; but so link albumen can make the structure of albumen change; and consumption wealth can be taken time and effort; bother very much; especially the reaction times of covalency link reaches a few hours; even several days, therefore the havoc activity of protein drug, twisted the practical function of protein drug.The present invention has prepared a kind of new nonlinear compound containing metal ion structure, utilize and there is the nonlinear new compound of the compound framework of branched structure, new texture dative bond and non covalent bond combine the relevant to be screened albumen having proved potential therapeutic action in vitro, coordinate animal model to screen the albumen having potential therapeutic activity quickly and easily in vivo.Should the feature in screening protein process of new nonlinear compound containing metal ion structure comprise: one, fast and simple, for screening in body is active and the protein-bonded used time is less than 15 minutes; Two, in body, screening process does not change protein structure, and covalency Ligature does not occur for this screening process and albumen.
Summary of the invention
1. its structure is as follows:
Wherein A representation metal ion, preferred calcium ion, magnesium ion, cobalt ion, barium ion, platinum ion, nickel ion, cupric ion.Molecular weight polyethylene glycol wherein in polymkeric substance is the integer between 100-200000, m=1-5000, the integer between preferred m=1-2000.The preparation method of this compound, is characterized in that:
1) the structure A containing nitrilotriacetic acid(NTA)-Methionin and compd B are obtained by reacting Compound C in sodium hydrogen carbonate solution;
2) Compound C obtains compd E with containing after the Y type Compound D stirring reaction of polyoxyethylene glycol; Molecular weight polyethylene glycol wherein in polymkeric substance is 100-200000;
3) compd E and metal ion compound react to obtain compound F 17-hydroxy-corticosterone;
Compd A;
Compd B;
Compound C;
Compound D, the integer between m=1-5000, the integer between preferred m=1-2000;
Compd E;
End product F, A representation metal ion.Wherein said chemical step selects solvent to be selected from: one or more in acetic acid, formic acid, benzene, toluene, pyridine, tetrahydrofuran (THF), trifluoroacetic acid, chloroform, tetracol phenixin, methylene dichloride, methyl alcohol, ethanol, methylene dichloride, dimethyl sulfoxide (DMSO), DMF.
Wherein most preferred configuration is:
With
Integer between m=1-5000, the integer between preferred m=1-2000.
Compound of the present invention makes the purposes of using at fast and simple screening albumen.This compound protein-bonded used time in screening process is less than 15 minutes, and this compound adopts non covalent bond associated proteins.The muscle, vein or the preparation of topical that are suitable for screening in body can be prepared into after this compound dative bond associated proteins.It can be ordinary preparation, controlled release preparation, targeting preparation etc.The wherein said chemical step of whole reaction selects solvent to be selected from: one or more in acetic acid, formic acid, benzene, toluene, pyridine, tetrahydrofuran (THF), trifluoroacetic acid, chloroform, tetracol phenixin, methylene dichloride, methyl alcohol, ethanol, methylene dichloride, dimethyl sulfoxide (DMSO), DMF.
Preparation method of the present invention is specific as follows:
By compd A (being purchased) 10mg-10g and compd B (being purchased) 0.01g-5g as 0.1-96 hour in solvent, 50 degree of-100 degree heating cooling after 1-48 hour, regulate PH to 2-5, after concentrating under reduced pressure, wash in ethanol, obtain Compound C after drying, the Y type Compound D (being purchased) containing polyoxyethylene glycol and the C that are purchased stir 1-10 hour in phosphoric acid salt, concentrating under reduced pressure obtains compd E, and the metal ion compound of compd E and 1-5mol reacts 1-48 hour to obtain final product compound F 17-hydroxy-corticosterone.F simply mixes 1-15 minute with equimolar target protein subsequently, the freeze-dried preparation then directly using or namely obtain after making related preparations freeze-drying.
Reaction scheme is:
Compd A
+
Compd B
Generate
Compound C;
Compound C
+
Compound D
Generate
Compd E;
Compd E
+
Metal ion compound
Generate
Compound F 17-hydroxy-corticosterone
Compound F 17-hydroxy-corticosterone with simply mix 15 minutes with histidine-tagged albumen and obtain compound F 17-hydroxy-corticosterone coordination associated proteins, and it to be directly used in body screening or being prepared into related preparations is used for screening in body.
Wherein compound F 17-hydroxy-corticosterone with histidine-tagged protein bound mode See Figure:
The end product prepared at this patent can screen the albumen proving to have potential therapeutic action in vitro in fast convenient and time-saving ground in vivo.
?
accompanying drawing illustrates:
fig. 1the nuclear magnetic resonance map preparing product of embodiment 1.
fig. 2the nuclear magnetic resonance map preparing product of embodiment 2.
Embodiment
Specific embodiment is described in further detail the present invention below, but the present invention not only limits to following examples.
Preparation embodiment is as follows:
embodiment 1
Compd A (being purchased) 500mg is dissolved in 6ml water, with 600mg sodium bicarbonate and 500mg compd B (being purchased) as in same beaker 15 hours, cooling after heating 70 degree, acetic acid regulator solution PH to 3, after concentrating under reduced pressure, wash in ethanol, crystallization obtains Compound C, excess ethanol washs rear drying again, the Y type Compound D (being purchased) that 400mg contains polyoxyethylene glycol is dissolved in the phosphoric acid buffer of PH7.5, add 2ml methyl alcohol, and stir 2 hours with Compound C, concentrating under reduced pressure, washing with alcohol, filter and wash again, vacuum-drying obtains compd E, the CoCl of compd E and 2Mol
2react to obtain compound F 17-hydroxy-corticosterone.
embodiment 2
Compd A (being purchased) 1000mg is dissolved in 6ml water, with 700mg sodium bicarbonate and 1100mg compd B (being purchased) as in same beaker 24 hours, cooling after heating 75 degree, acetic acid regulator solution PH to 3.3, after concentrating under reduced pressure, wash in ethanol, crystallization obtains Compound C, excess ethanol washs rear drying again, the Y type Compound D (being purchased) that 800mg contains polyoxyethylene glycol is dissolved in the phosphoric acid buffer of PH7.3, add 3ml dimethyl sulfoxide (DMSO), and stir 2 hours with Compound C, concentrating under reduced pressure, washing with alcohol, filter and wash again, vacuum-drying obtains compd E, the CaCl of compd E and 3Mol
2deserved compound F 17-hydroxy-corticosterone in the aqueous solution of a small amount of methylene dichloride.
effect experimental is as follows:
Citing TRAIL(TNF-related apoptosis-inducing ligand) albumen, its preparation method reference: S. Y. Chae, T. H. Kim, K. Park, C. H. Jin, S. Son, S. Lee, Y. S. Youn, K. Kim, D. G. Jo, I. C. Kwon, X. Chen, K. C. Lee, Mol. Cancer Ther. 2010, 9, 1719-1729, the potential albumen of in-vitro screening is replaced with TRAIL, go to combine with compound of the present invention and compare the advantage of the compounds of this invention in vivo in efficient and convenient screening process, the trail protein ended up containing Histidine of the sample prepared by embodiment 1-2 and equimolar amount simply mixes 15 minutes in aqueous.Sample grouping is specially:
First group is embodiment 1 sample sets coordination TRAIL, and the trail protein of 20mgF and 0.1mg/ml prepared by Example 1 is simple in the phosphoric acid buffer of PH7.4 mixes 15 minutes;
Second group is embodiment 2 sample sets coordination TRAIL, and the trail protein of 20mgF and 0.1mg/ml prepared by Example 1 is simple in the phosphoric acid buffer of PH7.4 mixes 10 minutes;
3rd group is polyoxyethylene glycol covalency link-group, and namely TRAIL-PEG, TRAIL and polyoxyethylene glycol generation covalency link (24 hours consuming time of whole link process);
4th group is polyoxyethylene glycol, metal ion simply mixes with TRAIL (30 minutes consuming time);
5th group is TRAIL group (naked albumen);
effect experimental is as follows:
Each group of sample is used for the treatment of H22 tumor-bearing mice situation compare.
Preparation H22 liver cancer mouse model, conventionally, implants mouse oxter by H22 cell, after preparation H22 core knurl mouse model, is divided into 6 groups, i.e. control group (not medication) and each group of medication,
First group is embodiment 1 sample sets coordination TRAIL, and the trail protein of 20mgF and 0.1mg/ml prepared by Example 1 simply mixes then freeze-drying in 15 minutes in the phosphoric acid buffer of PH7.4, and to weigh up the molten water of 10mg stand-by.
Second group is embodiment 2 sample sets coordination TRAIL, and the trail protein of 20mgF and 0.1mg/ml prepared by Example 1 simply mixes then freeze-drying in 10 minutes in the phosphoric acid buffer of PH7.4, and to weigh up the molten water of 10mg stand-by.
3rd group is polyoxyethylene glycol covalency link-group, and namely TRAIL-PEG, TRAIL and polyoxyethylene glycol generation covalency link (24 hours consuming time of whole link process), takes out 10mg and is dissolved in water stand-by.
4th group is polyoxyethylene glycol 0.2mol/ml, metallic ions Ca Cl
20.2mol/ml and TRAIL0.2mol/ml simply mixes (30 minutes consuming time) in phosphoric acid buffer, weighs up the molten water of 10mg stand-by.
5th group is TRAIL group (naked albumen), weighs up the molten water of 10mg stand-by.
Enter in knurl body by each group of Sample Injection, often group injection 50ul, concentration is 1mg/ml, often organizes 20 mouse.After testing 2 weeks, put to death mouse, get knurl kind of calliper volume, result is as follows.
Table 1 knurl size compares
Table 1 gross tumor volume (unit: cm3)
| Group | Knurl size |
| Control group | 14.12 1.77 |
| First group | 4.88 0.76** |
| Second group | 4.92 0.84** |
| 3rd group | 12.07 1.12* |
| 4th group | 13.87 1.45 |
| 5th group | 14.21 1.49 |
* p<0.05 is compared, * * p<0.01 with control group,
Can find out from experimental data, compound of the present invention can being less than in 15 minutes, under the condition not changing protein structure, non covalent bond associated proteins also screens its activity in vivo, make originally can not in vivo very well the protein expression of show activity gone out extraordinary activity, can find out thus, the albumen showing potential treatment ability in vitro can not be screened activity with omitting by the bright compound of we and screening method well in animal body, for clinical experiment future this albumen being applied in human body provides the experiment basis entered by force, protein drug formulation for future is provided possible selection by this technology simultaneously.
Claims (10)
1. the new compound structure be shown below, its structure is as follows:
Wherein A representation metal ion, preferred calcium ion, magnesium ion, cobalt ion, barium ion, platinum ion, nickel ion, cupric ion.
2. the polymkeric substance of claim 1, the molecular weight polyethylene glycol wherein in polymkeric substance is the integer between 100-200000, m=1-5000, the integer between preferred m=1-2000.
3. the preparation method of compound as claimed in claim 1, is characterized in that:
1) the structure A containing nitrilotriacetic acid(NTA)-Methionin and compd B are obtained by reacting Compound C in sodium hydrogen carbonate solution;
2) Compound C obtains compd E with containing after the Y type Compound D stirring reaction of polyoxyethylene glycol; Molecular weight polyethylene glycol wherein in polymkeric substance is 100-200000;
3) compd E and metal ion compound react to obtain compound F 17-hydroxy-corticosterone;
Compd A;
Compd B;
Compound C;
Compound D, the integer between m=1-5000, the integer between preferred m=1-2000;
Compd E;
End product F, A representation metal ion.
4. the method for claim 3, wherein said chemical step selects solvent to be selected from: one or more in acetic acid, formic acid, benzene, toluene, pyridine, tetrahydrofuran (THF), trifluoroacetic acid, chloroform, tetracol phenixin, methylene dichloride, methyl alcohol, ethanol, methylene dichloride, dimethyl sulfoxide (DMSO), DMF.
5. the compound of claim 1, most preferred configuration is
With
Integer between m=1-5000, the integer between preferred m=1-2000.
6. the compound of claim 1, described compound makes the purposes of using at fast and simple screening albumen.
7. the purposes of claim 6, this compound protein-bonded used time in screening process is less than 15 minutes.
8. the purposes of claim 6, this compound adopts non covalent bond associated proteins.
9. the muscle, vein or the preparation of topical that are suitable for screening in body after the compound dative bond associated proteins of claim 1, can be prepared into.
10. the described preparation of claim 9, it can be ordinary preparation, controlled release preparation, targeting preparation etc.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104211948A (en) * | 2013-06-03 | 2014-12-17 | 韩文毅 | Novel nonlinear polymer, and preparation method and use thereof |
| CN104650343A (en) * | 2013-06-03 | 2015-05-27 | 韩文毅 | Novel non-linear polymer as well as preparation method and application thereof |
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| CN1829711A (en) * | 2003-04-11 | 2006-09-06 | 格兰马克药品股份有限公司 | Novel heterocyclic compounds useful for the treatment of inflammatory and allergic disorders: process for their preparation and pharmaceutical compositions containing them |
| WO2006138350A2 (en) * | 2005-06-15 | 2006-12-28 | Anormed Inc. | Chemokine receptor binding compounds |
| WO2010008864A2 (en) * | 2008-06-24 | 2010-01-21 | Amira Pharmaceuticals, Inc. | Cycloalkane[b]indole angtagonists of prostaglandin d2 receptors |
| CN101928318A (en) * | 2009-06-24 | 2010-12-29 | 天津金耀集团有限公司 | Preparation of fluorine-containing steroid hormone |
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2013
- 2013-05-31 CN CN201310212661.6A patent/CN104211947A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1829711A (en) * | 2003-04-11 | 2006-09-06 | 格兰马克药品股份有限公司 | Novel heterocyclic compounds useful for the treatment of inflammatory and allergic disorders: process for their preparation and pharmaceutical compositions containing them |
| WO2006138350A2 (en) * | 2005-06-15 | 2006-12-28 | Anormed Inc. | Chemokine receptor binding compounds |
| WO2010008864A2 (en) * | 2008-06-24 | 2010-01-21 | Amira Pharmaceuticals, Inc. | Cycloalkane[b]indole angtagonists of prostaglandin d2 receptors |
| CN101928318A (en) * | 2009-06-24 | 2010-12-29 | 天津金耀集团有限公司 | Preparation of fluorine-containing steroid hormone |
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| SHARON WAICHMAN，ET AL.: "Maleimide Photolithography for Single-Molecule Protein-Protein Interaction Analysis in Micropatterns", 《ANALYTICAL CHEMISTRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104211948A (en) * | 2013-06-03 | 2014-12-17 | 韩文毅 | Novel nonlinear polymer, and preparation method and use thereof |
| CN104650343A (en) * | 2013-06-03 | 2015-05-27 | 韩文毅 | Novel non-linear polymer as well as preparation method and application thereof |
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Application publication date: 20141217 |
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