CN101240026A - Conjugate constructed from normal tridecyl and RGD peptide, and its synthesis and application in medicine - Google Patents

Conjugate constructed from normal tridecyl and RGD peptide, and its synthesis and application in medicine Download PDF

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CN101240026A
CN101240026A CNA2007100636676A CN200710063667A CN101240026A CN 101240026 A CN101240026 A CN 101240026A CN A2007100636676 A CNA2007100636676 A CN A2007100636676A CN 200710063667 A CN200710063667 A CN 200710063667A CN 101240026 A CN101240026 A CN 101240026A
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CN101240026B (en
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彭师奇
赵明
崔国辉
蒋雪云
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Capital Medical University
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Abstract

The invention discloses a target cationic pharmacosome with n-tridecyl and RGD peptide as basic component, and further composed by carbonyl, methylamino and methoxy. Preparation and application is also disclosed. The inventive target cationic pharmcosome has a structure of general formula CH<SUB>3</SUB>(CH<SUB>2</SUB>)<SUB>12</SUB>R<SUB>1</SUB>, wherein R<SUB>1</SUB> is carbonyl, methylamino or RGD pipetide modified by methoxy. The inventive target cationic pharmacosome has excellent target property and self-assembly property besides excellent vein and oral antithrombotic activity, can be used as preparation material or target material for preparation of drug carrier such as microemulsion, liposomes.

Description

The conjugate that n-tridecane base and RGD peptide make up, their the synthetic and application in medical science
Technical field
The present invention relates to n-tridecane base chain and put together the target cationic pharmacome that gets by the carboxylic end or the ammonia end of carbonyl, methylamino-and methoxyl group and RGD peptide, the invention still further relates to the preparation method of this pharmacome and this pharmacome as the application of antithrombotic agent, pharmaceutical carrier or drug targeting preparation material, belong to biomedicine field.
Background technology
Gene therapies in 5 years to 10 years in future have the ideal carrier that can be sent to gene cell.From the position of biotechnological formulation, the gene transfer system is exactly drug delivery system (DDS).The gene transfer system that has number of chemical synthetic small molecules to make up receives publicity, and wherein the gene transfer system of cation lipid formation is the focus of attention.Cation lipid at pharmaceutical field all over being used to make up liposomal delivery small molecules (comprising polypeptide) medicine.With the same in DDS, cationic-liposome has obvious benefit in the gene transfer system, for example can by electric charge absorption form mixture with DNA and effectively avoid by lysosome degraded in the cell, can with the efficient transfered cell of DNA, to the size of DNA without limits, easy and simple to handle etc.The essential structure of cation lipid is as follows:
Figure A20071006366700031
The essential structure of cation lipid
Comprise hydrophobic arm and hydrophilic cationic head that senior aliphatic chain constitutes.
Cell adhesion plays a crucial role in the evolution of cell adhesion disease (metastasis of cancer, thrombosis, chemistry cause inflammation and osteoporosis).Modulability glycoprotein for example RGD peptide and integrin receptor has very strong binding ability, can participate in the cell adhesion process.For example the RGD peptide combines with the GP IIb/IIIa receptor-specific of thrombocyte, tumour cell and bone primary surface and can intervene thrombus, metastasis of cancer and osteoporotic evolution.This effect of RGD peptide has given the compound that contains the RGD sequence a kind of critical nature, and the compound that promptly contains the RGD sequence can be to thrombus, metastasis of cancer and osteoporotic disease sites enrichment.Under such prerequisite, RGD peptide and senior aliphatic chain are puted together, just can obtain the target pharmacome.
Summary of the invention
One of the object of the invention provides a class target cationic pharmacome.This class target cationic pharmacome is general formula CH 3(CH 2) 12R 1Shown in, R wherein 1Be selected from CORGDS, CORGDV, CORGDF, RGDSOCH 2, RGDVOCH 2, RGDFOCH 2, RGDSNHCH 2, RGDVNHCH 2Or RGDFNHCH 2Wherein said R is the abbreviation of arginine (Arg), and G is the abbreviation of glycine (Gly), and D is the abbreviation of aspartic acid (Asp), and V is the abbreviation of Xie Ansuan (Val), and F is the abbreviation of phenylalanine (Phe), and S is the abbreviation of Serine (Ser).
The present invention combines following understanding or foundation has been finished technique scheme.The ideal carrier (gene transfer system) that gene is sent to cell is one of mission critical of gene therapy; See that from the position of biotechnological formulation gene transfer system and drug delivery system (DDS) have identity property; The gene transfer system that cation lipid constitutes though the gene transfer system that has number of chemical synthetic small molecules to make up all receives publicity should especially pay close attention; Cationic-liposome can form mixture with DNA by electric charge absorption and also effectively avoid by lysosome degraded in the cell; Cationic-liposome can be with the efficient transfered cell of DNA, cationic-liposome to the size of DNA without limits; The compound that contains the RGD sequence can be to thrombus, inflammation, metastasis of cancer and osteoporotic disease sites enrichment.
The inventor is based on above-mentioned cognition, n-tridecane base chain is puted together by the carboxylic end or the ammonia end of carbonyl, methylamino-and methoxyl group and RGD peptide, make the molecule that makes obtain four kinds of performances, promptly depend on the cationic property and the wetting ability of the hydrophobicity of n-tridecane base chain, the guanidine radicals that depends on the RGD peptide and the protonated formation of alpha-amino group and depend on the target that the RGD peptide obtains GP IIb/IIIa receptor-specific affinity interaction.So, RGD peptide and n-tridecane base chain are puted together the molecule that makes and just become the target cationic pharmacome.
One of purpose of the present invention provides a kind of preparation general formula CH 3(CH 2) 12R 1The method of compound, wherein R 1Be selected from-CORGDS ,-CORGDV ,-CORGDF, RGDSOCH 2-, RGDVOCH 2-, RGDFOCH 2-, RGDSNHCH 2-, RGDVNHCH 2-or RGDFNHCH 2-.
One of purpose of the present invention is achieved through the following technical solutions:
(1) meets peptide technology synthetic respectively RGDS, RGDV and RGDF according to existing liquid phase;
(2) with CH 3(CH 2) 12COOH, CH 3(CH 2) 12CH 2OH and CH 3(CH 2) 12CH 2NH 2With RGDS, RGDV and the RGDF coupling of protecting group protection, slough protecting group, promptly respectively.
The evaluation that forms on the model at rat suppository shows that target cationic pharmacome of the present invention has outstanding vein and oral antithrombotic acitivity, can be used as antithrombotic agent and uses; Detected these 9 kinds of target cationic pharmacomes at water and biophase and be self-assembled into performance into nano particle, test-results shows that target cationic pharmacome of the present invention has outstanding self-assembly performance, can be used as the preparation material of preparation micro emulsion, liposome etc.; Because the vein of these 9 kinds of target cationic pharmacomes and oral antithrombotic acitivity from the RGD peptide to GP IIb/IIIa receptor-specific affinity interaction, so this antithrombotic acitivity has characterized the target of 9 kinds of target cationic pharmacomes again, so target cationic pharmacome of the present invention can be used as the targeting preparation material.
Description of drawings
Fig. 1 RGD peptide and 14 aliphatic chains are puted together the synthetic route of preparation target cationic pharmacome.I) DCC, HOBt, NMM and HClGlyOMe; II) the NaOH aqueous solution (2N); III) DCC, HOBt, NMM and HClSer-OBzl or HClVal-OBzl or HClPhe-OBzl; IV) hydrogenchloride/ethyl acetate solution (4N); V) DCC, HOBt, NMM; VI) 5% palladium carbon and hydrogen (0.02Mba). AA=Phe wherein, Val, Ser.
The nanometer ball that Fig. 2 is assembled at water by the representative compounds of the present invention that scanning electron microscope is described.
The nanometer ball that Fig. 3 is assembled at fat mutually by the representative compounds of the present invention that scanning electron microscope is described.
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and not should be understood to limitation of the present invention.
Embodiment
The preparation of embodiment 1 RGDS
1) Boc-Arg (NO 2The preparation of)-Gly-OMe
With 1.600g (5.0mmol) Boc-Arg (NO 2)-OH is dissolved in the 20ml dry DMF, and ice bath adds 0.675g (5mmol) N-hydroxy benzo triazole (HOBt) down in the solution that obtains, and makes dissolving fully.Add 1.071g (6mmol) dicyclohexyl carbonyl diimine (DCC) after 10 minutes.Obtain reaction solution (I), stand-by.The following 0.627g of ice bath (5.0mmol) HClGly-OMe (5mmol) is suspended in the 20ml dry DMF, adds 1ml N-methylmorpholine (NMM) then, transfers pH 8-9.Stirred 35 minutes, and obtained reaction solution (II), stand-by.The following reaction solution of ice bath (I) adds in the reaction solution (II), and first ice bath stirs 1h down, stirring at room 12h again, and TLC (chloroform/methanol, 10: 1) shows Boc-Arg (NO 2)-OH disappears.Filtering dicyclohexylurea (DCU) (DCU), filtrate is blown away DMF.Residue dissolves with the 50ml chloroform.The solution that obtains is used saturated NaHCO successively 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed, 5%KHSO 4The aqueous solution is washed with the saturated NaCl aqueous solution and is washed.The chloroform layer anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, obtain 3.80g (100%) title compound, are the beige solid.ESI-MS(m/z)392[M+H] +。[α] D 20＝-2.1967
2) Boc-Arg (NO 2The preparation of)-Gly-OH
With 0.392g (1.0mmol) Boc-Arg (NO 2)-Gly-OMe is dissolved in 10ml methyl alcohol.Under the ice bath solution that obtains is transferred pH12 and stirred 2h with NaOH (2N) aqueous solution, TLC (chloroform/methanol, 1: 1) shows Boc-Arg (NO 2)-Gly-OMe disappears.Reaction mixture is transferred pH7 with dilute hydrochloric acid (2N), and concentrating under reduced pressure removes methyl alcohol.Residue is transferred pH2, is used ethyl acetate extraction (30ml * 3) with dilute hydrochloric acid (2N).The ethyl acetate that merges is washed till neutrality, anhydrous Na with the saturated NaCl aqueous solution mutually 2SO 4Dry.Filter, filtrate decompression is concentrated into dried, gets 0.339g (90%) title compound, is the beige solid.ESI-MS(m/z)377[M+H] +。[α] D 20＝-1.4813(c＝1.0，CH 3OH)。
3) preparation of Boc-Asp (OBzl)-Ser (Bzl)-OBzl
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe gets 2.931g (99%) title compound by 1.620g (5.0mmol) Boc-Asp (OBzl)-OH and 1.605g (5.0mmol) TosSer (Bzl)-OBzl, is beige oily body.ESI-MS(m/z)591[M+H] +。[α] D 20＝-13.2108(c＝1.0，CHCl 3)
4) preparation of HClAsp (OBzl)-Ser (Bzl)-OBzl
0.780g (1.491mmol) Boc-Asp (OBzl)-Ser (Bzl)-OBzl is dissolved in 15ml 4mol/l hydrogenchloride-ethyl acetate solution, stirring at room 2 hours, TLC (chloroform/methanol, 3/1) shows that raw material point disappears, concentrating under reduced pressure is removed ethyl acetate, and residue adds a small amount of ether repeatedly and carries out concentrating under reduced pressure to remove hydrogen chloride gas.Add a small amount of ether at last residue is ground to form 0.570g (95.98%) title compound, be pressed powder, be directly used in next step reaction.ESI-MS(m/z)491[M+H] +。[α] D 20＝-55.4369(c＝1.0，CH 3OH)。
5) Boc-Arg (NO 2The preparation of)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 0.831g (3.0mmol) Boc-Arg (NO 2)-Gly-OH and 1.470g (3.0mmol) HClAsp (OBzl)-Ser (Bzl)-OBzl gets 2.931g (99%) title compound, is the beige solid.ESI-MS(m/z)851[M+H] +。[α] D 20=-16.3729 (c=1.0, CHCl 3) 6) HClArg (NO 2The preparation of)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.850g (1.0mmol) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl makes 0.744g (99.2%) title compound, is the beige solid.ESI-MS(m/z)751[M+H] +。[α] D 20＝-58.3196(c＝1.0，CH 3OH)。
7) preparation of RGDS
With 0.750g (1.0mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl places the 50ml eggplant-shape bottle, with dissolve with ethanol, add 35mgPd/C (5%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried, and residue grinds with sherwood oil repeatedly, get 405mg (90%) title compound, are the colorless solid powder.ESI-MS(m/z)450[M+H] +。[α] 20 D＝6.0(c＝1.0，H 2O).
The preparation of embodiment 2RGDV
1) preparation of Boc-Asp (OBzl)-Val-OBzl
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe gets 2.5344g (99%) title compound by 1.620g (5.0mmol) Boc-Asp (OBzl)-OH and 1.895g (5.0mmol) TosVal-OBzl, is the beige solid.
ESI-MS(m/z)513[M+H] +。[α] D 20=-14.2710 (c=0.2, chloroforms).
2) preparation of HClAsp (OBzl)-Val-OBzl
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, get 0.662g (99%) title compound from 0.763g (1.491mmol) Boc-Asp (OBzl)-Val-OBzl, be pressed powder, be directly used in next step reaction.MS (M+1:413.4) [α] D 20=-47.8027 (c=0.2, chloroforms).
3) Boc-Arg (NO 2The preparation of)-Gly-Asp (OBzl)-Val-OBzl
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 0.831g (3.0mmol) Boc-Arg (NO 2)-Gly-OH and 1.3455g (3.0mmol) HClAsp (OBzl)-Val-OBzl gets 2.08g (90%) title compound, is the beige solid.ESI-MS(m/z)772[M+H] +。[α] D 20=-30.9346 (c=0.2, chloroforms).
4) HClArg (NO 2The preparation of)-Gly-Asp (OBzl)-Val-OBzl
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.771g (1.0mmol) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Val-OBzl makes 0.637g (95%) title compound, is the beige solid.ESI-MS(m/z)672[M+H] +。[α] D 20=-50.8685 (c=0.2, chloroforms).
5) preparation of RGDV
According to the preparation method of RGDS, from 671mg (1.0mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Val-OBzl gets 405mg (91%) title compound, is the colorless solid powder.The 99mg target compound.ESI-MS(m/z)446[M+H] +。[α] 20 D＝3.0(c＝1.0，H 2O).
The preparation of embodiment 3RGDF
1) preparation of Boc-Asp (OBzl)-Val-OBzl
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe gets 2.77g (90%) title compound by 1.620g (5.0mmol) Boc-Asp (OBzl)-OH and 2.135g (5.0mmol) TosPhe-OBzl, is the beige solid.
ESI-MS(m/z)561[M+H] +。[α] D 20=-15.2842 (c=0.2, chloroforms).
2) preparation of HClAsp (OBzl)-Phe-OBzl
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, get 0.703g (95%) title compound from 0.835g (1.491mmol) Boc-Asp (OBzl)-Phe-OBzl, be pressed powder, be directly used in next step reaction.ESI-MS(m/z)461[M+H] +。[α] D 20=-44.8753 (c=0.2, chloroforms).
3) Boc-Arg (NO 2The preparation of)-Gly-Asp (OBzl)-Phe-OBzl
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 0.831g (3.0mmol) Boc-Arg (NO 2)-Gly-OH and 1.4895g (3.0mmol) HClAsp (OBzl)-Phe-OBzl gets 2.16g (88%) title compound, is the beige solid.ESI-MS(m/z)820[M+H] +。[α] D 20=-20.3582 (c=0.2, chloroforms).
4) HClArg (NO 2The preparation of)-Gly-Asp (OBzl)-Phe-OBzl
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.819g (1.0mmol) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Phe-OBzl makes 0.717g (95%) title compound, is the beige solid.ESI-MS(m/z)719[M+H] +。[α] D 20=-10.6121 (c=0.2, chloroforms).
5) preparation of RGDF
According to the preparation method of RGDS, from 718mg (1.0mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Phe-OBzl gets 454mg (92%) title compound, is the colorless solid powder.ESI-MS(m/z)494[M+H] +。[α] 20 D＝4.0(c＝2.0，H 2O).
Embodiment 4CH 3(CH 2) 12The preparation of CORGDS
1) CH 3(CH 2) 12CO-Arg (NO 2The preparation of)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.12g (5.0mmol) CH 3(CH 2) 12CO 2H and 3.750g (5.0mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl makes 4.108g (85.6%) title compound, is yellow waxy solid.ESI-MS(m/z)961[M+H] +。[α] D 20=-10.2810 (c=0.2, chloroforms).
2) CH 3(CH 2) 12The preparation of CO-Arg-Gly-Asp-Ser-OH
According to the preparation method of RGDS by 0.960g (1mmol) CH 3(CH 2) 12CO-Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl makes 0.421g (64%) title compound, is yellow powder shape solid.ESI-MS(m/z)645[M+H] +。[α] D 20=-4.0953 (c=0.2, chloroforms).
Embodiment 5CH 3(CH 2) 12The preparation of CORGDV
1) CH 3(CH 2) 12CO-Arg (NO 2The preparation of)-Gly-Asp (OBzl)-Val-OBzl
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.12g (5.0mmol) CH 3(CH 2) 12CO 2H and 3.350g (5.0mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Val-OBzl makes 3.612g (82%) title compound, is yellow waxy solid.ESI-MS(m/z)881[M+H] +。[α] D 20=-6.0237 (c=0.2, chloroforms).
2) CH 3(CH 2) 12The preparation of CO-Arg-Gly-Asp-Val-OH
According to the preparation method of RGDS by 0.881g (1mmol) CH 3(CH 2) 12CO-Arg (NO 2)-Gly-Asp (OBzl)-Val-OBzl makes 0.546g (83%) title compound, is yellow solid.ESI-MS(m/z)656[M+H] +。[α] D 20=-32.9926 (c=0.2, chloroforms).
Embodiment 6CH 3(CH 2) 12The preparation of CORGDF
1) CH 3(CH 2) 12CO-Arg (NO 2The preparation of)-Gly-Asp (OBzl)-Phe-OBzl
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.12g (5.0mmol) CH 3(CH 2) 12CO 2H and 3.59g (5.0mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Phe-OBzl makes 3.39g (73%) title compound, is yellow waxy solid.ESI-MS(m/z)930[M+H] +。[α] D 20=-9.8210 (c=0.2, chloroforms).
2) CH 3(CH 2) 12The preparation of CO-Arg-Gly-Asp-Phe-OH
According to the preparation method of RGDS by 0.929g (1mmol) CH 3(CH 2) 12CO-Arg (NO 2)-Gly-Asp (OBzl)-Phe-OBzl makes 0.633g (90%) title compound, is yellow solid.ESI-MS(m/z)705[M+H] +。[α] D 20=-18.5103 (c=0.2, chloroforms).
Embodiment 76) RGDS-OCH 2(CH 2) 12CH 3Preparation
1) Boc-Ser (BZl)-OCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.48g (5mmol) Boc-Ser (BZl)-OH and 1.07g (5.0mmol) CH 3(CH 2) 12CH 2OH makes 2.41g (98%) title compound, is the colorless oil body.
ESI-MS(m/z)493[M+H] +。[α] D 20=9.5044 (c=0.2, chloroforms).
2) HClSer (Bzl)-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.491g (1.0mmol) Boc-Ser (BZl)-OCH 2(CH 2) 12CH 3Make 0.371g (93.2%) title compound, be the beige solid.
ESI-MS(m/z)393[M+H] +。[α] D 20=-1.0989 (c=0.2, chloroforms).
3) Boc-Asp (OBzl)-Ser (Bzl)-OCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.615g (5mmol) Boc-Asp (OBzl)-OH and 1.955g (5.0mmol) HClSer (Bzl)-OCH 2(CH 2) 12CH 3Make 3.132g (90%) title compound, be the yellow oily body.ESI-MS(m/z)670[M+H] +。[α] D 20=-42.7341 (c=0.2, chloroforms).
4) HClAsp (OBzl)-Ser (Bzl)-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.696g (1.0mmol) Boc-Asp (OBzl)-Ser (BZl) O 2CH 2(CH 2) 12CH 3Make 0.574g (96.3%) title compound, be the beige solid.ESI-MS(m/z)570[M+H] +。[α] D 20=-55.4369 (c=0.2, chloroforms).
5) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Ser (BZl)-OCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.88g (5.0mmol) Boc-Arg (NO 2)-Gly-OH and 2.98g (5.0mmol) HClAsp (OBzl)-Ser (Bzl)-OCH 2(CH 2) 12CH 3Make 4.05g (85%) title compound, be yellow solid.ESI-MS(m/z)956[M+H] +。[α] D 20=-36.1891 (c=0.2, chloroforms).
6) HClArg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.955g (1.0mmol) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OCH 2(CH 2) 12CH 3Make 0.832g (97.8%) title compound, be the beige solid.ESI-MS(m/z)856[M+H] +。[α] D 20＝-25.1762
7) RGDS-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of RGDS by 0.880g (1mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OCH 2(CH 2) 12CH 3Make 0.526g (83.6%) title compound, be colorless solid.ESI-MS(m/z)630[M+H] +。[α] D 20=-8.1905 (c=0.2, chloroforms).
Embodiment 8RGDV-OCH 2(CH 2) 12CH 3Preparation
1) Boc-Val-OCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.08g (5mmol) Boc-Val-OH and 1.07g (5.0mmol) CH 3(CH 2) 12CH 2OH makes 3.98g (96.4%) title compound, is the colorless oil body.ESI-MS(m/z)415[M+H] +。[α] D 20=-5.0246 (c=0.2, chloroforms).
2) HClVal-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.413g (1.0mmol) Boc-Val-OCH 2(CH 2) 12CH 3Make 0.325g (90.7%) title compound, be beige oily body.ESI-MS(m/z)314[M+H] +。[α] D 20=-18.5711 (c=0.2, chloroforms).
3) Boc-Asp (OBzl)-Val-OCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.615g (5mmol) Boc-Asp (OBzl)-OH and 1.747g (5.0mmol) HClVal-OCH 2(CH 2) 12CH 3Make 2.592g (83.9%) title compound, be the yellow oily body.ESI-MS(m/z)620[M+H] +。[α] D 20=-14.8418 (c=0.2, chloroforms).
4) HClAsp (OBzl)-Val-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.618g (1.0mmol) Boc-Asp (OBzl)-Val-OCH 2(CH 2) 12CH 3Make 0.538g (97%) title compound, be the beige solid.ESI-MS(m/z)520[M+H] +。[α] D 20=-11.4835 (c=0.2, chloroforms).
5) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Val-OCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.88g (5.0mmol) Boc-Arg (NO 2)-Gly-OH and 2.77g (5.0mmol) HClAsp (OBzl)-Val-OCH 2(CH 2) 12CH 3Make 3.33g (76%) title compound, be yellow solid.ESI-MS(m/z)878[M+H] +。[α] D 20=-19.8647 (c=0.2, chloroforms).
6) HClArg (NO 2)-Gly-Asp (OBzl)-Val-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.877g (1.0mmol) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Val-OCH 2(CH 2) 12CH 3Make O.785g (96.5%) title compound, be the beige solid.ESI-MS(m/z)778[M+H] +。[α] D 20=-13.1467 (c=0.2, chloroforms).
7) RGDV-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of RGDS by 0.813g (1mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Val-OCH 2(CH 2) 12CH 3Make 0.516g (80.4%) title compound, be colorless solid.ESI-MS(m/z)642[M+H] +。[α] D 20=-9.8539 (c=0.2, chloroforms).
Embodiment 9RGDF-OCH 2(CH 2) 12CH 3Preparation
1) Boc-Phe-OCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.32g (5mmol) Boc-Phe-OH and 1.07g (5.0mmol) CH 3(CH 2) 12CH 2OH makes 2.093g (91%) title compound, is the yellow oily body.ESI-MS(m/z)462[M+H] +。[α] D 20=-26.4765 (c=0.2, chloroforms).
2) HClPhe-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.460g (1.0mmol) Boc-Phe-OCH 2(CH 2) 12CH 3Make 0.357g (90%) title compound, be the beige solid.ESI-MS(m/z)362[M+H] +。[α] D 20=-12.9836 (c=0.2, chloroforms).
3) Boc-Asp (OBzl)-Phe-OCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.62g (5mmol) Boc-Asp (OBzl)-OH and 2.0g (5.0mmol) HClPhe-OCH 2(CH 2) 12CH 3((81.2%) title compound is the yellow oily body to make 2.697g.ESI-MS(m/z)667[M+H] +。[α] D 20=-33.2249 (c=0.2, chloroforms).
4) HClAsp (OBzl)-Phe-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.667g (1.0mmol) Boc-Asp (OBzl)-Phe-OCH 2(CH 2) 12CH 3Make 0.543g (90%) title compound, be the beige solid.ESI-MS(m/z)567[M+H] +。[α] D 20=-31.8653 (c=0.2, chloroforms).
5) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Phe-OCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.88g (5.0mmol) Boc-Arg (NO 2)-Gly-OH and 3.02g (5.0mmol) HClAsp (OBzl)-Phe-OCH 2(CH 2) 12CH 3Make 3.42g (74%) title compound, be yellow solid.ESI-MS(m/z)925[M+H] +。
[α] D 20=-37.6554 (c=0.2, chloroforms).
6) HClArg (NO 2)-Gly-Asp (OBzl)-Phe-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, 0.925g (1.0mmol) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Phe-OCH 2(CH 2) 12CH 3Make 0.854g (80%) title compound, be the beige solid.ESI-MS(m/z)826[M+H] +。[α] D 20=-16.3284 (c=0.2, chloroforms).
7) RGDF-OCH 2(CH 2) 12CH 3Preparation
According to the preparation method of RGDS by 0.861g (1mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Phe-OCH 2(CH 2) 12CH 3Make? g (90%) title compound is colorless solid.ESI-MS(m/z)690[M+H] +。[α] D 20=-9.4877 (c=0.2, chloroforms).
Embodiment 10RGDS-NHCH 2(CH 2) 12CH 3Preparation
1) Boc-Ser (BZl)-NHCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.48g (5mmol) Boc-Ser (BZl)-OH and 1.07g (5.0mmol) CH 3(CH 2) 12CH 2NH 2Make 2.41g (98%) title compound, for being the beige solid.ESI-MS(m/z)491[M+H] +。[α] D 20=-8.4230 (c=0.2, chloroforms).
2) HClSer (BZl)-NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.490g (1.0mmol) Boc-Ser (BZl)-NH 2CH 2(CH 2) 12CH 3Make 0.424g (95%) title compound, be the beige solid.ESI-MS(m/z)391[M+H] +。[α] D 20=-17.7019 (c=0.2, chloroforms).
3) Boc-Asp (OBzl)-Ser (BZl) NHCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.62g (5mmol) Boc-Asp (OBzl)-OH and 2.13g (5.0mmol) CH 3(CH 2) 12CH 2NH 2Make 2.81g (81%) title compound, be the beige solid.
ESI-MS(m/z)696[M+H] +。[α] D 20=-21.5863 (c=0.2, chloroforms).
4) HClAsp (OBzl)-Ser (BZl) NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.695g (1.0mmol) Boc-Asp (OBzl)-Ser (BZl) NHCH 2(CH 2) 12CH 3Make 0.58g (92%) title compound, be the beige solid.ESI-MS(m/z)596[M+H] +。[α] D 20=-23.3851 (c=0.2, chloroforms).
5) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Ser (BZl) NHCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.88g (5.0mmol) Boc-Arg (NO 2)-Gly-OH and 3.15g (5.0mmol) HClAsp (OBzl)-Ser (BZl) NHCH 2(CH 2) 12CH 3Make 3.816g (80%) title compound, be yellow solid.ESI-MS(m/z)955[M+H] +。[α] D 20=-43.2821 (c=0.2, chloroforms).
6) HClArg (NO 2)-Gly-Asp (OBzl)-Ser (BZl) NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.954g (1.0mmol) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Ser (BZl) NHCH 2(CH 2) 12CH 3Make 0.756g (85%) title compound, be the beige solid.ESI-MS(m/z)855[M+H] +。[α] D 20=-16.1643 (c=0.2, chloroforms).
7) RGDS-NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of RGDS by 0.890g (1.0mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Ser (BZl) O 2CH 2(CH 2) 12CH 3Make 0.616g (83.4%) title compound, be colorless solid.ESI-MS(m/z)630[M+H] +。[α] D 20=-6.0595 (c=0.2, chloroforms).
Embodiment 11RGDV-NHCH 2(CH 2) 12CH 3Preparation
1) Boc-Val-NHCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.08g (5mmol) Boc-Val-OH and 1.07g (5.0mmol) CH 3(CH 2) 12CH 2NH 2Make 1.85g (90%) title compound, be colorless solid.
ESI-MS(m/z)414[M+H] +。[α] D 20=-4.162 (c=0.2, chloroforms).
2) HClVal-NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.412g (1.0mmol) Boc-Val-NHCH 2(CH 2) 12CH 3Make 0.296g (85%) title compound, be the beige solid.ESI-MS(m/z)314[M+H] +。[α] D 20=-12.6192 (c=0.2, chloroforms).
3) Boc-Asp (OBzl)-Val-NHCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.67g (5mmol) Boc-Asp (OBzl)-OH and 1.74g (5.0mmol) HClVal-NHCH 2(CH 2) 12CH 3Make 2.468g (80%) title compound, be the beige solid.ESI-MS(m/z)618[M+H] +。[α] D 20=-79.1534 (c=0.2, chloroforms).
4) HClAsp (OBzl)-Val-NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.617g (1.0mmol) Boc-Asp (OBzl)-Val-NHCH 2(CH 2) 12CH 3Make 0.498g (90%) title compound, be the beige solid.ESI-MS(m/z)518[M+H] +。[α] D 20=-36.6215 (c=0.2, chloroforms).
5) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Val-NHCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.88g (5.0mmol) Boc-Arg (NO 2)-Gly-OH and 2.77g (5.0mmol) HClAsp (OBzl)-Val-NHCH 2(CH 2) 12CH 3Make 3.28g (75%) title compound, be the beige solid.ESI-MS(m/z)899[M+H] +。[α] D 20＝-25.6373
6) HClArg (NO 2)-Gly-Asp (OBzl)-Val-NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.876g (1.0mmol) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Val-NHCH 2(CH 2) 12CH 3Make 0.690g (85%) title compound, be the beige solid.ESI-MS(m/z)776[M+H] +。[α] D 20=-19.3158 (c=0.2, chloroforms).
7) RGDV-NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of RGDS by 0.812g (1mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Val-NHCH 2(CH 2) 12CH 3Make 0.480g (75%) title compound, be the beige solid.ESI-MS(m/z)642[M+H] +。[α] D 20=-36.2917 (c=0.2, chloroforms).
Embodiment 12RGDF-NHCH 2(CH 2) 12CH 3Preparation
1) Boc-Phe-NHCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.32g (5mmol) Boc-Phe-OH and 1.07g (5.0mmol) CH 3(CH 2) 12CH 2NH makes 1.95g (85%) title compound, is the beige solid.ESI-MS(m/z)462[M+H] +。[α] D 20=-2.6303 (c=0.2, chloroforms).
2) HClPhe-NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.36g (1.0mmol) Boc-Phe-NHCH 2(CH 2) 12CH 3Make 0.356g (90%) title compound, be the beige solid.ESI-MS(m/z)361[M+H] +。[α] D 20=6.9228 (c=0.2, chloroforms).
3) Boc-Asp (OBzl)-Phe-NHCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.62g (5mmol) Boc-Asp (OBzl)-OH and 1.98g (5.0mmol) HClPhe-NHCH 2(CH 2) 12CH 3Make 2.66g (80%) title compound, be the beige solid.ESI-MS(m/z)667[M+H] +。[α] D 20=-5.0589 (c=0.2, chloroforms).
4) HClAsp (OBzl)-Phe-NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.665g (1.0mmol) Boc-Asp (OBzl)-Phe-NHCH 2(CH 2) 12CH 3Make 0.542g (90%) title compound, be the beige solid.ESI-MS(m/z)567[M+H] +。[α] D 20=-3.6549 (c=0.2, chloroforms).
5) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Phe-NHCH 2(CH 2) 12CH 3Preparation
According to Boc-Arg (NO 2The preparation method of)-Gly-OMe is by 1.88g (5.0mmol) Boc-Arg (NO 2)-Gly-OH and 3.01g (5.0mmol) HClAsp (OBzl)-Phe-NHCH 2(CH 2) 12CH 3Make 3.47g (75%) title compound, be yellow solid.ESI-MS(m/z)926[M+H] +。[α] D 20=-3.7440 (c=0.2, chloroforms).
6) HClArg (NO 2)-Gly-Asp (OBzl)-Phe-NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of HClAsp (OBzl)-Ser (Bzl)-OBzl, from 0.924g (1.0mmol) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Phe-NHCH 2(CH 2) 12CH 3Make 0.774g (90%) title compound, be the beige solid.ESI-MS(m/z)826[M+H] +。[α] D 20=-13.6491 (c=0.2, chloroforms).
7) RGDF-NHCH 2(CH 2) 12CH 3Preparation
According to the preparation method of RGDS by 0.861g (1mmol) HClArg (NO 2)-Gly-Asp (OBzl)-Phe-OCH 2(CH 2) 12CH 3Make 0.522g (75%) title compound, be colorless solid.ESI-MS(m/z)690[M+H] +。[α] D 20=-31.9939 (c=0.2, chloroforms).
The antithrombotic acitivity test of test example 1 The compounds of this invention intravenously administrable
1) rat operation and apparatus
(male, 220～230g) press 1200mg-kg to the SD rat -1Dosage abdominal injection urethane solution is anaesthetized.The anesthetized rat dorsal position is fixed, and separates right common carotid artery, and in proximal part folder bulldog clamp, proximal part and distal end penetrate surgical thread respectively, and the surgical thread of distal end is clamped with mosquito forceps in fur, prepares in the distal end intubate.
2) intubate
Intubate is the polyethylene rubber tube that silanization is crossed, and divides three sections, and the stage casing is a polyethylene rubber tube, long 60.0mm, internal diameter 3.5mm; Two ends are identical polyethylene tube, and pipe range 100.0mm, internal diameter 1.0mm, an end of this pipe of external diameter 2.0mm pull into point pipe (being used to insert rat carotid artery or vein), and external diameter is 1.0mm.Be respectively charged into the long black surgical thread of 6cm in the clean penicillin bottle with the number of finishing, weigh; Take out silk thread then, put into the thicker intubate in stage casing of ready intubate according to numbering.
Open rat right side bulldog clamp, will fill with heparin-saline solution (50IUkg in the pipe by sharp pipe end with syringe -1), then the arterial end of intubate is inserted the rat right carotid, the heparin of calculated amount is slowly injected in the rat body.
3) administration
Medicine: physiological saline (3mlkg -1), the normal saline solution of Asprin (dosage is 30mg/kg), the normal saline solution of The compounds of this invention (dosage is 5 μ mol/kg).
Folder closes rat right carotid artery folder, pulls up the syringe of intubate vein end, has the water-soluble medical fluid injector of medicine of calculated amount to insert the vein end of intubate suction, opens rat right carotid artery folder, and medicine is slowly pushed in the rat body.Folder closes right carotid artery folder, and the vein end in teahouse is inserted the rats with left jugular vein of separator well, opens bulldog clamp, makes blood begin circulation.And pick up counting simultaneously.Can produce thrombus because of blood circulation on the silk thread in this process in the extra heavy pipe of intubate central authorities.
4) thrombus is weighed.
Timing is cut off venous incubation after beginning 15 minutes, stops circulation, carefully takes out silk thread with the ophthalmology tweezer, dips in drop of blood on filter paper gently, puts into the penicillin bottle of weighing in advance, accurately weighs and record.Calculate the weight in wet base of thrombus.Each medicine repeats 11 administrations.The wet weight of thrombus of each group of statistics (X ± SD), and do the t check.
5) result
Through intravenously administrable, compound of the present invention all has good anti-thrombus activity.The result lists table 1 in.
Table 1 compound of the present invention is through the antithrombotic acitivity of intravenously administrable
Compound Wet weight of thrombus (X ± SDmg)
Physiological saline 20.97±3.18
Asprin 10.10±3.01 a
CH 3(CH 2) 12CORGDS 12.07±5.34 a
CH 3(CH 2) 12CORGDV 11.77±5.05 a
CH 3(CH 2) 12CORGDF 11.50±3.38 a
RGDS-OCH 2(CH 2) 12CH 3 11.85±5.05 a
RGDV-OCH 2(CH 2) 12CH 3 15.54±4.14 a
RGDF-OCH 2(CH 2) 12CH 3 15.82±5.81 a
RGDS-NHCH 2(CH 2) 12CH 3 11.65±5.78 a
RGDV-NHCH 2(CH 2) 12CH 3 15.54±4.14 a
RGDF-NHCH 2(CH 2) 12CH 3 7.78±2.93 a
N=11; A. compare P＜0.01 with physiological saline.
The antithrombotic acitivity test of test example 2 The compounds of this invention oral administrations
1) rat operation and apparatus
(male, 220～230g) press 10nmolkg to the SD rat -1Oral dose compound of the present invention is pressed 1200mg-kg behind the 30min -1Dosage abdominal injection urethane solution is anaesthetized.The anesthetized rat dorsal position is fixed, and separates right common carotid artery, and in proximal part folder bulldog clamp, proximal part and distal end penetrate surgical thread respectively, and the surgical thread of distal end is clamped with mosquito forceps in fur, prepares in the distal end intubate.
2) intubate
Intubate is the polyethylene rubber tube that silanization is crossed, and divides three sections, and the stage casing is a polyethylene rubber tube, long 60.0mm, internal diameter 3.5mm; Two ends are identical polyethylene tube, and pipe range 100.0mm, internal diameter 1.0mm, an end of this pipe of external diameter 2.0mm pull into point pipe (being used to insert rat carotid artery or vein), and external diameter is 1.0mm.Be respectively charged into the long black surgical thread of 6cm in the clean penicillin bottle with the number of finishing, weigh; Take out silk thread then, put into the thicker intubate in stage casing of ready intubate according to numbering.
Open rat right side bulldog clamp, will fill with heparin-saline solution (50IUkg in the pipe by sharp pipe end with syringe -1), then the arterial end of intubate is inserted the rat right carotid, the heparin of calculated amount is slowly injected in the rat body.
3) give drug solns
Medicine: physiological saline (3mlkg -1, orally give), the normal saline solution, the normal saline solution of The compounds of this invention (dosage is 10nmol/kg, orally give) of Asprin (dosage is 30mg/kg, gives by the method vein of test example 1).
4) thrombus is weighed
Timing is cut off venous incubation after beginning 15 minutes, stops circulation, carefully takes out silk thread with the ophthalmology tweezer, dips in drop of blood on filter paper gently, puts into the penicillin bottle of weighing in advance, accurately weighs and record.Calculate the weight in wet base of thrombus.Each medicine repeats 11 administrations.The wet weight of thrombus of each group of statistics (X ± SD), and do the t check.
5) result
The oral administration administration, compound of the present invention all has good anti-thrombus activity.The result lists table 2 in.
The antithrombotic acitivity of table 2. compound oral administration of the present invention administration
Compound Wet weight of thrombus (X ± SDmg)
Physiological saline 28.80±1.40
Asprin 8.50±0.70 a
CH 3(CH 2) 12CORGDS 8.92±3.93 a
CH 3(CH 2) 12CORGDV 8.73±3.88 a
CH 3(CH 2) 12CORGDF 10.98±3.12 a
RGDS-OCH 2(CH 2) 12CH 3 12.81±3.79 a
RGDV-OCH 2(CH 2) 12CH 3 10.15±3.26 a
RGDF-OCH 2(CH 2) 12CH 3 15.65±3.47 a
RGDS-NHCH 2(CH 2) 12CH 3 6.94±3.18 a
RGDV-NHCH 2(CH 2) 12CH 3 6.87±2.55 a
RGDF-NHCH 2(CH 2) 12CH 3 9.04±2.02 a
N=10; A. compare P＜0.01 with physiological saline.
Test example 3 The compounds of this invention CH 3(CH 2) 12The dose-effect relationship of CORGDS oral administration
1) rat operation and apparatus
(male, 220～230g) press 10nmolkg to the SD rat -1, 0.1nmolkg -1And 0.001nmolkg -1Oral dose CH 3(CH 2) 12CORGDS presses 1200mg-kg behind the 30min -1Dosage abdominal injection urethane solution is anaesthetized.The anesthetized rat dorsal position is fixed, and separates right common carotid artery, and in proximal part folder bulldog clamp, proximal part and distal end penetrate surgical thread respectively, and the surgical thread of distal end is clamped with mosquito forceps in fur, prepares in the distal end intubate.
2) intubate
Intubate is the polyethylene rubber tube that silanization is crossed, and divides three sections, and the stage casing is a polyethylene rubber tube, long 60.0mm, internal diameter 3.5mm; Two ends are identical polyethylene tube, and pipe range 100.0mm, internal diameter 1.0mm, an end of this pipe of external diameter 2.0mm pull into point pipe (being used to insert rat carotid artery or vein), and external diameter is 1.0mm.Be respectively charged into the long black surgical thread of 6cm in the clean penicillin bottle with the number of finishing, weigh; Take out silk thread then, put into the thicker intubate in stage casing of ready intubate according to numbering.
Open rat right side bulldog clamp, will fill with heparin-saline solution (50IUkg in the pipe by sharp pipe end with syringe -1), then the arterial end of intubate is inserted the rat right carotid, the heparin of calculated amount is slowly injected in the rat body.
3) give drug solns
Medicine: with CH 3(CH 2) 10CORGDS presses 10nmolkg -1, 0.1nmolkg -1And 0.001nmolkg -1Dosage configuration normal saline solution, for oral administration.
4) thrombus is weighed
Timing is cut off venous incubation after beginning 15 minutes, stops circulation, carefully takes out silk thread with the ophthalmology tweezer, dips in drop of blood on filter paper gently, puts into the penicillin bottle of weighing in advance, accurately weighs and record.Calculate the weight in wet base of thrombus.Each medicine repeats 11 administrations.The wet weight of thrombus of each group of statistics (X ± SD), and do the t check.
5) result
The oral administration administration is at 10nmolkg -1, 0.1nmolkg -1, 0.001nmolkg -1And 0.0001nmolkg -1Under the dosage, CH 3(CH 2) 12CORGDS dosage relies on ground performance anti thrombotic action.The result lists table 3 in.
Table 3. orally give CH 3(CH 2) 12The dose-effect relationship of CORGDS
Dosage Wet weight of thrombus (X ± SDmg)
10nmol/Kg/3ml 6.87±2.55 a
0.1nmol/Kg/3ml 12.83±3.37 b
0.001nmol/Kg/3ml 13.19±2.72 b
0.0001nmol/Kg/3ml 19.10±3.33
N=10; A. with 0.1nmolkg -1, 0.001nmolkg -1And 0.0001nmolkg -1The group ratio, p＜0.05; B. with 0.001nmolkg -1And 0.0001nmolkg -1The group ratio, p＜0.05.
Test example 4 The compounds of this invention are in the test of aqueous phase assembling becoming nanometer ball
1) 9 kinds of positively charged ion pharmacomes of the present invention are configured as the aqueous solution according to the concentration of mg/ml, go up METHOD FOR CONTINUOUS DETERMINATION 8 days at laser light scattering particle size analyzer (model), observe particle diameter and change of size, the results are shown in Table 4.The data that obtain show that these 9 kinds of positively charged ion pharmacomes can be self-assembled at aqueous phase and be the stabilized nano ball, thereby are the outstanding micro emulsion and the preparation material of liposome medicament.
Table 49 kinds of positively charged ion pharmacomes of the present invention are self-assembled into the grain warp into the stabilized nano ball in water
The sample name The 1st day The 2nd day The 3rd day The 4th day The 5th day The 6th day The 7th day The 8th day
RGDF-OCH 2(CH 2) 12CH 3 77.31 134.3 140 135.8 151.4 170.4 173.5 175.8
RGDV-OCH 2(CH 2) 12CH 3 125 176.7 211.1 227.6 257.4 128.5 777.6 454
RGDS-OCH 2(CH 2) 12CH 3 67.3 80.08 100.7 101.5 115 276.4 139.4 178.3
RGDF-NHCH 2(CH 2) 12CH 3 82.83 94.94 108.3 109.4 119.2 115.1 120.1 137.1
RGDV-NHCH 2(CH 2) 12CH 3 62.4 124.2 179.3 209.1 266.9 280.3 320.8 315
RGDS-NHCH 2(CH 2) 12CH 3 48.85 144.3 209.4 185.6 417.6 360 889.9 1946
CH 3(CH 2) 12CORGDF 198.8 202.9 243.2 334.2 217 259.9 247.7 247
CH 3(CH 2) 12CORGDV 62.57 501.9 249.2 245.4 324.1 274.6 275.9 256.8
CH 3(CH 2) 12CORGDS 2738 3057 1240 2193 725.3 1214 1031 1581
2) according to the mensuration requirement of scanning electron microscope, the aqueous solution of 9 kinds of positively charged ion pharmacomes of configuration places the dry back of copper mesh Back stroke to measure the figure of compound of the present invention at the nanometer ball of water assembling.The result shows, can see uniform granular attitude by the nanometer ball of these 9 kinds of positively charged ion pharmacome assemblings under scanning electron microscope.Given here is the sem photograph (Fig. 2) of representative compounds.
Test example 5 compounds of the present invention fat mutually in assembling become nanometer ball and test
1) selecting n-Octanol is the fat phase, and 9 kinds of positively charged ion pharmacomes of the present invention are configured as n-Octanol solution according to the concentration of mg/ml, goes up METHOD FOR CONTINUOUS DETERMINATION 8 days at laser light scattering particle size analyzer (model), observes particle diameter and change of size, the results are shown in Table 5.The data that obtain show, these 9 kinds of positively charged ion pharmacomes can be self-assembled in mutually at fat and be the stabilized nano ball, thereby are the outstanding micro emulsion and the preparation material of liposome medicament.
Table 59 kinds of positively charged ion pharmacomes of the present invention are self-assembled into the grain warp into the stabilized nano ball in n-Octanol
The sample name The 1st day The 3rd day The 8th day
RGDF-OCH 2(CH 2) 12CH 3 541.3nm 757.4nm 695.9nm
RGDV-OCH 2(CH 2) 12CH 3 1421nm 1675nm 1388nm
RGDS-OCH 2(CH 2) 12CH 3 793.5nm 859.6nm 1085nm
RGDF-NHCH 2(CH 2) 12CH 3 1057nm 1071nm 1003nm
RGDV-NHCH 2(CH 2) 12CH 3 1738nm 2184nm 1121nm
RGDS-NHCH 2(CH 2) 12CH 3 914.8nm 1195nm 1358nm
CH 3(CH 2) 12CORGDF 855.1nm 6773nm 4496nm
CH 3(CH 2) 12CORGDV 1554nm 2481nm 2296nm
CH 3(CH 2) 12CORGDS 1457nm 2384nm 2857nm
2) according to the mensuration requirement of scanning electron microscope, the n-Octanol solution of 9 kinds of positively charged ion pharmacomes of configuration places the dry back of copper mesh Back stroke to measure the figure of the nanometer ball that compound of the present invention assembles mutually at fat.The result shows, can see uniform granular attitude by the nanometer ball of these 9 kinds of positively charged ion pharmacome assemblings under scanning electron microscope.Given here is the sem photograph (Fig. 3) of representative compounds.

Claims (5)

1. the target cationic pharmacome is general formula CH 3(CH 2) 12R 1Shown in, R wherein 1Be selected from CORGDS, CORGDV, CORGDF, RGDSOCH 2, RGDVOCH 2, RGDFOCH 2, RGDSNHCH 2, RGDVNHCH 2Or RGDFNHCH 2
2. method for preparing claim 1 target cationic pharmacome comprises:
(1) meets peptide technology synthetic respectively RGDS, RGDV and RGDF according to conventional liquid phase;
(2) with CH 3(CH 2) 12COOH, CH 3(CH 2) 12CH 2OH and CH 3(CH 2) 12CH 2NH 2With RGDS, RGDV and the RGDF coupling of protecting group protection, slough protecting group, promptly respectively.
3. the target cationic pharmacome of claim 1 is in the purposes of preparation in the antithrombotic reagent.
4. the target cationic pharmacome of claim 1 prepares the purposes in the preparation material of micro emulsion or lipidosome drug carrier.
5. the target cationic pharmacome of claim 1 prepares the purposes in the targeting preparation material of micro emulsion, lipidosome drug carrier.
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