CN104211949A - Nonlinear polymer and preparation and use thereof - Google Patents
Nonlinear polymer and preparation and use thereof Download PDFInfo
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- CN104211949A CN104211949A CN201310218700.3A CN201310218700A CN104211949A CN 104211949 A CN104211949 A CN 104211949A CN 201310218700 A CN201310218700 A CN 201310218700A CN 104211949 A CN104211949 A CN 104211949A
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Abstract
The invention discloses a nonlinear polymeric compound and a preparation method and use of the nonlinear polymeric compound. The new nonlinear polymeric compound is constructed by use of a compound with the branched structure, and the non covalent bond of the new structure is combined with related to-be-in-vivo-screened protein with potential therapeutic effect for cooperating in vivo with an animal model for rapid and convenient screening of the protein with potential therapeutic activity. The nonlinear polymeric compound with a metal ion structure has the following characteristics in the protein screening process: 1, the nonlinear polymeric compound is quick and easy, and the time for bonding with the protein for in-vivo activity screening is less than 15 minutes; and 2, the in vivo screening process does not change the structure of the protein, and covalent linking reaction between the screening process and the protein may not occur.
Description
Technical field
The invention discloses preparation method and the purposes of a kind of 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, and the short shortcomings that waits of transformation period is determined in vitro it and potentially cannot effectively be screened in vivo activity after effectively.A lot of of great value protein drugs cannot be verified in vivo.There is scholar to have after the covalently bound albumen of compound of provide protection for screening in body or treatment with some; but so linking albumen can make the structure of albumen change; and can take time and effort consumption wealth; bother very much; especially the reaction times of covalency link reaches a few hours; even several days, thus havoc the activity of protein drug, twisted the practical function of protein drug.The present invention has prepared a kind of nonlinear polymerizable compound that contains metal ion structure, utilization have nonlinear organization compound framework nonlinear new compound, new texture dative bond is non covalent bond in conjunction with the relevant to be screened albumen of potential therapeutic action that proved in vitro, coordinates in vivo animal model to screen quickly and easily the albumen that has potential therapeutic activity.The feature of this nonlinear polymerizable compound that contains metal ion structure in screening protein process comprises: one, and fast and simple, for the active protein-bonded used time of screening in body 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:
A representation metal ion wherein, preferred calcium ion, magnesium ion, cobalt ion, barium ion, platinum ion, nickel ion, cupric ion.Wherein the molecular weight polyethylene glycol in polymkeric substance is 100-200000, the integer between m=1-5000, the preferably integer between m=1-2000.The preparation method of this compound, is characterized in that:
1) the structure A that contains nitrilotriacetic acid(NTA)-Methionin reacts and obtains Compound C with compd B in sodium hydrogen carbonate solution;
2) Compound C with containing obtaining compd E after the star-like compound D stirring reaction of polyoxyethylene glycol; Wherein the molecular weight polyethylene glycol in polymkeric substance is 100-200000;
3) compd E reacts to obtain compound F 17-hydroxy-corticosterone with metal ion compound;
Compd A;
Compd B;
Compound C;
Compound D, the integer between m=1-5000, the preferably integer between m=1-2000;
Compd E;
Reaction gained compound F 17-hydroxy-corticosterone, wherein 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 preferably integer between m=1-2000.
Compound of the present invention is made 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 in conjunction with albumen.This compound dative bond can be prepared into the muscle that is suitable for screening in body, vein or the preparation of topical after in conjunction with albumen.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 for 0.1-96 hour in solvent, cooling after 1-48 hour in 50 degree-100 degree heating, regulate PH to 2-5, after concentrating under reduced pressure, in ethanol, wash, after dry, obtain Compound C, the star-like compound D(that contains polyoxyethylene glycol being purchased is purchased) in phosphoric acid salt, stir 1-10 hour with C, concentrating under reduced pressure obtains compd E, and compd E reacts 1-48 hour to obtain final product compound F 17-hydroxy-corticosterone with the metal ion compound of 1-5mol.F simply mixes 1-15 minute with the target protein of 4 times of molar weights subsequently, then directly uses or make the freeze-dried preparation obtaining after 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 and albumen with histidine-tagged is simple to be mixed and within 15 minutes, obtains compound F 17-hydroxy-corticosterone coordination and be combined albumen, and it is directly used for screening in body or being prepared into related preparations uses for screening in body.
Wherein compound F 17-hydroxy-corticosterone with histidine-tagged protein bound mode, see following formula:
The end product of preparing at this patent can fast convenient and time-saving ground screening prove the albumen that has potential therapeutic action in vivo in vitro.
accompanying drawing explanation:
fig. 1the nuclear magnetic resonance map of preparing product of embodiment 1.
fig. 2the nuclear magnetic resonance map of 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 Example 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, in ethanol, wash, crystallization obtains Compound C, filtering ethanol washs rear dry again, the star-like compound D(that 400mg contains polyoxyethylene glycol (molecular weight 10000) is purchased) be 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 8Mol
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, in ethanol, wash, crystallization obtains Compound C, filtering ethanol washs rear dry again, the star-like compound D(that 800mg contains polyoxyethylene glycol (molecular weight 2000) is purchased) be 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 MgCl of compd E and 8Mol
2deserved compound F 17-hydroxy-corticosterone in the aqueous solution of a small amount of methylene dichloride.
effect experiment is as follows:
TRAIL(TNF-related apoptosis-inducing ligand gives an example) albumen, from reagent company, buy with histidine-tagged TRAIL, in this experiment, TRAIL hypothesis is represented to unknown activity in vivo, but prove in vitro the albumen of lateral reactivity, for testing the effect of the compounds of this invention.Specific experiment is as follows: the potential albumen that replaces in-vitro screening with TRAIL, with compound of the present invention go in conjunction with and the compounds of this invention advantage in efficient and convenient screening process in vivo relatively, the trail protein that contains Histidine ending of sample prepared by embodiment 1-2 and 4 times of molar weights is simple in the aqueous solution mixes 15 minutes (specific practice is as follows).Sample grouping is specially:
First group is embodiment 1 sample sets coordination TRAIL, gets the F of 20mg of embodiment 1 preparation and the trail protein solution of the 15ml of 0.4mg/ml and simply mixes 15 minutes in the phosphoric acid buffer of PH7.4;
Second group is embodiment 2 sample sets coordination TRAIL, gets the F of 20mg of embodiment 1 preparation and the trail protein solution of the 15ml of 0.4mg/ml and simply mixes 15 minutes in the phosphoric acid buffer of PH7.4;
The 3rd group is polyoxyethylene glycol (molecular weight 10000) 0.2mol/ml, metal ion compound CoCl
20.8mol/ml simply mixes 15 minutes with the trail protein solution of the 15ml of 0.4mg/ml;
The 4th group is polyoxyethylene glycol (molecular weight 2000) 0.2mol/ml, metal ion compound MgCl
20.8mol/ml simply mixes 15 minutes with the trail protein solution of the 15ml of 0.4mg/ml;
The 5th group is TRAIL group (naked albumen);
experiment in animal body:
Each group sample is used for the treatment of to H22 tumor-bearing mice situation to be compared.
Preparation H22 liver cancer mouse model, according to traditional method, implants mouse oxter by H22 cell, after preparation H22 core knurl mouse model, is divided into 6 groups, i.e. each group of control group (not medication) and medication,
First group is embodiment 1 sample sets coordination TRAIL, gets that the 20mgF of embodiment 1 preparation and the trail protein of 0.4mg/ml are simple in the phosphoric acid buffer of PH7.4 to be mixed then freeze-drying in 15 minutes to weigh up the molten water of 10mg stand-by.
Second group is embodiment 2 sample sets coordination TRAIL, gets that the 20mgF of embodiment 1 preparation and the trail protein of 0.4mg/ml are simple in the phosphoric acid buffer of PH7.4 to be mixed then freeze-drying in 15 minutes to weigh up the molten water of 10mg stand-by.
The 3rd group is polyoxyethylene glycol (molecular weight 10000) 0.2mol/ml, metal ion compound CoCl
20.8mol/ml simply mixes (15 minutes consuming time) in phosphoric acid buffer with TRAIL0.4mol/ml, it is stand-by that freeze-drying weighs up the molten water of 10mg.
The 4th group is polyoxyethylene glycol (molecular weight 2000) 0.2mol/ml, metal ion compound MgCl
20.8mol/ml simply mixes (15 minutes consuming time) in phosphoric acid buffer with TRAIL0.4mol/ml, it is stand-by that freeze-drying weighs up the molten water of 10mg.
The 5th group is TRAIL group (naked albumen), and it is stand-by that freeze-drying weighs up the molten water of 10mg.
Each group sample is injected in knurl body, every group of injection 50ul, concentration is 1mg/ml, every group of 20 mouse.Test after 2 weeks, put to death mouse, get knurl kind of calliper volume, result is as follows.
Table 1 knurl size relatively
Table 1 gross tumor volume (unit: cm3)
| Group | Knurl size |
| Control group | 14.12±1.77 |
| First group | 4.31±1.29** |
| Second group | 4.44±1.14** |
| The 3rd group | 13.97±1.81 |
| The 4th group | 14.04±1.97 |
| The 5th group | 14.12±1.88 |
Compare * p<0.05 with control group, * * p<0.01,
Can from experimental data, find out, compound of the present invention can be less than in 15 minutes, do not changing under the condition of protein structure, non covalent bond is in conjunction with albumen and screen in vivo its activity, make originally the albumen of fine show activity in vivo show extraordinary activity, can find out thus, the compound that we are bright and screening method can not omitted the albumen that shows in vitro potential treatment ability is screened active well in animal body, for the clinical experiment that future this albumen is applied in to human body provides the experiment basis that enters by force, this technology provides possible selection by the pharmaceutical grade protein preparation for following simultaneously.
Claims (10)
1. the new compound structure being shown below, its structure is as follows:
A representation metal ion wherein, preferred calcium ion, magnesium ion, cobalt ion, barium ion, platinum ion, nickel ion, cupric ion.
2. the polymkeric substance of claim 1, wherein the molecular weight polyethylene glycol in polymkeric substance is 100-200000, the integer between m=1-5000, the preferably integer between m=1-2000.
3. the preparation method of compound as claimed in claim 1, is characterized in that:
1) the structure A that contains nitrilotriacetic acid(NTA)-Methionin reacts and obtains Compound C with compd B in sodium hydrogen carbonate solution;
2) Compound C with containing obtaining compd E after the star-like compound D stirring reaction of polyethylene glycol structures; Wherein the molecular weight polyethylene glycol in polymkeric substance is 100-200000;
3) compd E reacts to obtain compound F 17-hydroxy-corticosterone with metal ion compound;
Compd A;
Compd B;
Compound C;
Compound D, the integer between m=1-5000, the preferably integer between 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 preferably integer between m=1-2000.
6. the compound of claim 1, described compound is made the purposes of using in vivo 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 in conjunction with albumen screening.
9. the compound dative bond of claim 1 can be prepared into the muscle that is suitable for screening in body, vein or the preparation of topical after in conjunction with albumen.
10. the described preparation of claim 9, it can be ordinary preparation, controlled release preparation, targeting preparation etc.
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| CN201310218700.3A CN104211949A (en) | 2013-06-04 | 2013-06-04 | Nonlinear polymer and preparation and use thereof |
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| CN201310218700.3A CN104211949A (en) | 2013-06-04 | 2013-06-04 | Nonlinear polymer and preparation and use thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104211950A (en) * | 2013-06-04 | 2014-12-17 | 韩文毅 | Nonlinear polymer and preparation and use 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 |
| CN104211950A (en) * | 2013-06-04 | 2014-12-17 | 韩文毅 | Nonlinear polymer and preparation and use thereof |
-
2013
- 2013-06-04 CN CN201310218700.3A patent/CN104211949A/en active Pending
Patent Citations (5)
| 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 |
| CN104211950A (en) * | 2013-06-04 | 2014-12-17 | 韩文毅 | Nonlinear polymer and preparation and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| S. RAHIMA BENHABBOUR，ET AL: "In vitro and in vivo assessment of targeting lipid-based nanoparticles to the epidermal growth factor-receptor (EGFR) using a novel Heptameric ZEGFR domain", 《JOURNAL OF CONTROLLED RELEASE》 * |
| SHARON WAICHMAN，ET AL.: "Maleimide Photolithography for Single-Molecule Protein-Protein Interaction Analysis in Micropatterns", 《ANALYTICAL CHEMISTRY》 * |
Cited By (1)
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| CN104211950A (en) * | 2013-06-04 | 2014-12-17 | 韩文毅 | Nonlinear polymer and preparation and use thereof |
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