CN101906141B - Conjugates of one class of peptide chain and double fatty alcohol chains as well as preparation method and application thereof - Google Patents
Conjugates of one class of peptide chain and double fatty alcohol chains as well as preparation method and application thereof Download PDFInfo
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- CN101906141B CN101906141B CN2009100851544A CN200910085154A CN101906141B CN 101906141 B CN101906141 B CN 101906141B CN 2009100851544 A CN2009100851544 A CN 2009100851544A CN 200910085154 A CN200910085154 A CN 200910085154A CN 101906141 B CN101906141 B CN 101906141B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 51
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 29
- 125000000373 fatty alcohol group Chemical group 0.000 title abstract 3
- 230000002785 anti-thrombosis Effects 0.000 claims abstract description 10
- 239000002502 liposome Substances 0.000 claims abstract description 4
- 239000004530 micro-emulsion Substances 0.000 claims abstract description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 79
- 150000001875 compounds Chemical class 0.000 claims description 63
- 150000002191 fatty alcohols Chemical group 0.000 claims description 21
- 125000006239 protecting group Chemical group 0.000 claims description 17
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- 229910000041 hydrogen chloride Inorganic materials 0.000 claims description 15
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 15
- 230000008878 coupling Effects 0.000 claims description 12
- 238000010168 coupling process Methods 0.000 claims description 12
- 238000005859 coupling reaction Methods 0.000 claims description 12
- -1 fatty alcohol aspartate Chemical class 0.000 claims description 11
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 9
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- 238000000034 method Methods 0.000 claims description 7
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- 238000012360 testing method Methods 0.000 description 7
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses conjugates of one type of peptide chain and double fatty alcohol chains as well as a preparation method and application thereof. One Arg-Gly-Asp-Phe-Asp peptide chain and two fatty alcohol chains are conjugated to obtain the conjugates with the general formula of Arg-Gly-Asp-Phe-Asp[-OCH2(CH2)nCH3]-OCH2(CH2)nCH3, wherein n equals to 6, 8, 10 or 12. The conjugates not only have excellent antithrombotic activity after being taken orally, but also have excellent self-assembly performance, and can also be used as preparation materials for preparing micro emulsions, liposome and other drug carriers.
Description
Technical field
The present invention relates to the conjugate of a class peptide chain and double fatty alcohol chain, relate in particular to the conjugate of Arg-Gly-Asp-Phe-Asp peptide chain and two aliphatic alcohol chains, and its preparation method and application, biomedicine field belonged to.
Background technology
GP IIb/IIIa is fibrinogenic acceptor on the platelet membrane, and it exposes when thrombocyte is activated and presents activity, is combined with fibrin-specific, causes the formation of hematoblastic gathering and thrombus.The polypeptide that contains the Arg-Gly-Asp sequence can capture the GP IIb/IIIa acceptor of platelet surface, so can suppress platelet aggregation and thrombosis that scleroproein participates in.Bibliographical information Arg-Gly-Asp-Phe polypeptide has good anti thrombotic action.
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 Arg-Gly-Asp peptide and integrin receptor has very strong binding ability, can participate in the cell adhesion process.For example the Arg-Gly-Asp peptide is combined with the GP IIb/IIIa of thrombocyte, tumour cell and bone primary surface receptor-specific and can be intervened thrombus, metastasis of cancer and osteoporotic evolution.This effect of Arg-Gly-Asp peptide has given the compound that contains the Arg-Gly-Asp sequence a kind of critical nature, and the compound that namely contains the Arg-Gly-Asp sequence can be to thrombus, metastasis of cancer and osteoporotic disease sites enrichment.Under such prerequisite, by Asp and senior aliphatic chain coupling, just can obtain the conjugate of Arg-Gly-Asp-Phe-Asp and two aliphatic alcohol chains to the Arg-Gly-Asp-Phe peptide.
Summary of the invention
One of purpose of the present invention provides a class and has the peptide chain of anti thrombotic action and the conjugate of the hydrophobic arm of double fatty alcohol.
One of purpose of the present invention is achieved through the following technical solutions:
The conjugate of one class peptide chain and double fatty alcohol chain is that the general formula of described conjugate is with the conjugate of an Arg-Gly-Asp-Phe-Asp peptide chain and two hydrophobic arm couplings of Fatty Alcohol(C12-C14 and C12-C18)
Arg-Gly-Asp-Phe-Asp[-OCH
2(CH
2)
nCH
3]-OCH
2(CH
2)
nCH
3, n=6 wherein, 8,10 or 12.
Two of purpose of the present invention provides a kind of above-mentioned method with peptide chain and double fatty alcohol chain conjugate of antithrombotic acitivity for preparing.
Two of purpose of the present invention is achieved through the following technical solutions:
A kind of method for preparing the conjugate of described peptide chain and double fatty alcohol chain comprises the steps:
Aspartic acid and two Fatty Alcohol(C12-C14 and C12-C18) condensations of (1) N being held protecting group protect, described Fatty Alcohol(C12-C14 and C12-C18) is CH
3(CH
2)
nCH
2OH, n=6,8,10,12;
(2) slough N end protecting group and obtain the double fatty alcohol aspartate;
(3) according to existing liquid phase synthetic technology, the double fatty alcohol aspartate is progressively connect peptide chain and the two fatty chain alcohol conjugate that the synthetic protecting group of peptide is protected with the protection intermediate of phenylalanine, the protection intermediate of aspartic acid, protection intermediate and the arginic protection intermediate of glycine successively;
(4) slough successively the peptide chain of protecting group protection and C end protecting group and the N end protecting group of two fatty chain alcohol conjugates and obtain target compound.
Wherein said N end protecting group is blocking group commonly used when the N end of polypeptide is protected, and for example can be tertbutyloxycarbonyl (Boc); Described C end protecting group is blocking group commonly used when the C end of polypeptide is protected, and for example can be methoxyl group (OMe); Described Side chain protective group is blocking group commonly used when the polypeptide side chain is protected, and for example can be benzyloxy (OBzl), nitro (NO
2); The process of described liquid phase synthetic technology and described protection, condensation, deprotection is the conventional and known technology of this area.
This preparation method can summarize with the route of Fig. 1, and concrete, described method comprises:
(1) in the presence of DCC, HOBt, NMM and THF with the aspartic acid of tertbutyloxycarbonyl protection respectively with CH
3(CH
2)
7OH, CH
3(CH
2)
9OH, CH
3(CH
2)
11OH and CH
3(CH
2)
13The OH coupling;
(2) in hydrogenchloride/ethyl acetate solution, add the conjugate of step (1) gained, slough tertbutyloxycarbonyl;
(3) phenylalanine and step (2) the products therefrom coupling in the presence of DCC, HOBt, NMM and THF, tertbutyloxycarbonyl protected;
(4) in hydrogenchloride/ethyl acetate solution, add the conjugate of step (3) gained, slough tertbutyloxycarbonyl;
Aspartic acid and step (4) the products therefrom coupling of (5) in the presence of DCC, HOBt, NMM and THF, holding tertbutyloxycarbonyl, protection side chain benzyloxy to protect N;
(6) in hydrogenchloride/ethyl acetate solution, add the conjugate of step (5) gained, slough tertbutyloxycarbonyl;
The glycine coupling of (7) in the presence of DCC, HOBt, NMM and THF, N being held arginine and the methoxyl group of tertbutyloxycarbonyl protection, the protection of side chain nitro to protect;
(8) in the presence of 2NNaOH, (7) products therefrom is removed C and hold protecting group;
(9) in the presence of DCC, HOBt, NMM and THF with (6) and (8) products therefrom coupling;
(10) (9) products therefrom deprotection base is obtained compound shown in the general formula.
Evaluation on the rat suppository formation model shows, the conjugate of peptide chain of the present invention and double fatty alcohol chain has outstanding oral antithrombotic acitivity, can be used as antithrombotic agent and uses; Use laser nano dynamics instrument, TEM, SEM and detected these 4 kinds of conjugates and be self-assembled into performance into nanostructure, test-results shows, conjugate of the present invention has outstanding self-assembly performance, can be used as the preparation material of preparation micro emulsion, liposome etc.; Because the oral antithrombotic acitivity of these 4 kinds of peptide chains and double fatty alcohol chain conjugate from the Arg-Gly-Asp peptide to GPIIb/IIIa receptor-specific affinity interaction, so this antithrombotic acitivity has characterized again the targeting of 4 kinds of conjugates, so conjugate of the present invention can be used as the targeting preparation material.
Description of drawings
Fig. 1 is the synthetic route of the conjugate of Arg-Gly-Asp-Phe-Asp and double fatty alcohol chain building.i)DCC/HOBt/NMM；ii)HCl/EtOAc；iii)H
2，Pd/C；9a n＝6，9b n＝8，9c n＝10，9d n＝12。
Fig. 2 is that laser nano dynamics instrument is measured compound of the present invention in the size distribution of aqueous phase.
Fig. 3-Fig. 6 is respectively the compound 9a-9d of the present invention that described by transmission electron microscope at the water assembling morphology.
Fig. 7 is that the compound 9a of the present invention that described by scanning electron microscope is at the water assembling morphology.
Fig. 8 and Fig. 9 are that the compound 9b of the present invention that described by scanning electron microscope is at the water assembling morphology.
Figure 10 is that the compound 9c of the present invention that described by scanning electron microscope is at the water assembling morphology.
Figure 11 and Figure 12 are that the compound 9d of the present invention that described by scanning electron microscope is at the water assembling morphology.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
Embodiment 1Boc-Asp[-OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Preparation (1a)
In the 100ml eggplant-shape bottle, add 2.33g (0.01mol) Boc-Asp, after anhydrous THF dissolving, in reaction solution, add 2.97g (0.022mol) HOBt and 4.53g (0.022mol) DCC, add 2.86g (0.022mol) eight alcohol behind the condition of ice bath stirring reaction 0.5h, regulate between the pH8-9 with NMM again, after reaction is spent the night, TLC shows that Boc-Asp raw material point disappears, filtering reacting liquid is removed DCU, again filtrate decompression is concentrated into dried, with 100ml acetic acid ethyl dissolution residue, filter and again remove insoluble DCU, ethyl acetate layer is used 5% sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, 5% aqueous potassium hydrogen sulfate, saturated sodium-chloride water solution, saturated sodium bicarbonate aqueous solution, each extraction of saturated sodium-chloride water solution is washed three times, tells the ethyl acetate layer anhydrous Na
2SO
4Dried overnight is filtered, and filtrate decompression is concentrated into dried, gets 4.52 (yield 99%) title compound, the off-white color solid.ESI-MS(m/z)：480[M+Na]
+.
Embodiment 2Boc-Asp[-OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Preparation (1b)
According to the operation of 1a, with 2.33g (0.01mol) Boc-Asp and 3.47g (0.022mol) ten alcohol, make 5.08g (yield 99%) title compound, the off-white color solid.ESI-MS(m/z)：536[M+Na]
+.
Embodiment 3Boc-Asp[-OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Preparation (1c)
According to the operation of 1a, make 5.63g (yield 98%) title compound, off-white color solid with 2.33g (0.01mol) Boc-Asp and 4.09g (0.022mol) lauryl alcohol.ESI-MS(m/z)：592[M+Na]
+.
Embodiment 4Boc-Asp[-OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Preparation (1d)
According to the operation of 1a, with 2.33g (0.01mol) Boc-Asp and 4.71g (0.022mol) tetradecyl alcohol, make 6.19g (yield 99%) title compound, the off-white color solid.ESI-MS(m/z)：648[M+Na]
+.
Embodiment 5Asp[-OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Preparation (2a)
With a small amount of ethyl acetate 1a 4.52g (0.0099mol) is dissolved, ice bath stirs the lower 4N of adding HCl/ ethyl acetate solution to be mixed, and TLC shows that raw material point disappears, and the solution with water pump is drained repeatedly, the Ex-all hydrogen chloride gas gets 3.53g (yield 99%) target compound.ESI-MS(m/z)：358[M+H]
+.
Embodiment 6Asp[-OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Preparation (2b)
Operation according to 2a makes 4.13g (yield 99%) target compound with 5.08g (0.0099mol) 1b.ESI-MS(m/z)：414[M+H]
+.
Embodiment 7Asp[-OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Preparation (2c)
Operation according to 2a makes 4.59g (yield 99%) target compound with 5.63g (0.0099mol) 1c.ESI-MS(m/z)：470[M+H.]
+.
Embodiment 8Asp[-OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Preparation (2d)
Operation according to 2a makes 5.15g (yield 99%) target compound with 6.19g (0.0099mol) 1d.ESI-MS(m/z)：526[M+H]
+.
Embodiment 9Boc-Phe-Asp[-OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Preparation (3a)
In the 100ml eggplant-shape bottle, add 3.18g (0.012mol) Boc-Phe, after anhydrous THF dissolving, in reaction solution, add 1.48g (0.011mol) HOBt and 2.26g (0.011mol) DCC, (the N end should be first with regulating between the pH8-9 with NMM after the anhydrous THF dissolving again to add 3.53 (0.0098mol) 2a behind the condition of ice bath stirring reaction 0.5h, add in the reaction solution again), after reaction is spent the night, TLC shows that N end raw material point disappears, filtering reacting liquid is removed DCU, again filtrate decompression is concentrated into dried, with 100ml acetic acid ethyl dissolution residue, filter and again remove insoluble DCU, ethyl acetate layer is used 5% sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, 5% aqueous potassium hydrogen sulfate, saturated sodium-chloride water solution, saturated sodium bicarbonate aqueous solution, each extraction of saturated sodium-chloride water solution is washed three times, tells the ethyl acetate layer anhydrous Na
2SO
4Dried overnight is filtered, and filtrate decompression is concentrated into dried, gets 5.97g (yield 99%) title compound, the off-white color solid.Mp：68.8-69.3℃；
(c＝0.8，CHCl
3)；ESI-MS(m/z)：627[M+Na]
+。
Embodiment 10Boc-Phe-Asp[-OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Preparation (3b)
Operation according to 3a makes 6.41g (yield 99%) title compound, off-white color solid with 3.18g (0.012mol) Boc-Phe and 4.13g (0.0098mol) 2b.Mp：63.2-65.1℃；
(c＝1，CHCl
3)；ESI-MS(m/z)：683[M+Na]
+。
Embodiment 11Boc-Phe-Asp[-OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Preparation (3c)
Operation according to 3a makes 7.02g (yield 99%) title compound, white powder with 3.18g (0.012mol) Boc-Phe and 4.59g (0.0098mol) 2c.Mp：65.4-66.7℃；
(c＝1，CHCl
3)；ESI-MS(m/z)：739[M+Na]
+。
Embodiment 12Boc-Phe-Asp[-OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Preparation (3d)
Operation according to 3a makes 7.58g (yield 99%) title compound, white powder with 3.18g (0.012mol) Boc-Phe and 5.15g (0.0098mol) 2d.Mp：59.6-62.2℃；
(c＝1，CHCl
3)；ESI-MS(m/z)：795[M+Na]
+。
Embodiment 13Phe-Asp[-OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Preparation (4a)
With a small amount of ethyl acetate 5.97 (0.0098mol) 3a is dissolved, ice bath stirs the lower 4N of adding HCl/ ethyl acetate solution to be mixed, and TLC shows that raw material point disappears, and the solution with water pump is drained repeatedly, the Ex-all hydrogen chloride gas gets 4.89 yields 99%) target compound.ESI-MS(m/z)：505[M+H]
+.
Embodiment 14Phe-Asp[-OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Preparation (4b)
Operation according to 4a makes 5.49g (yield 99%) target compound with 6.41g (0.0098mol) 3b.ESI-MS(m/z)：561[M+H]
+.
Embodiment 15Phe-Asp[-OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Preparation (4c)
Operation according to 4a makes 6.04g (yield 99%) target compound with 7.02g (0.0098mol) 3c.ESI-MS(m/z)：617[M+H]
+.
Embodiment 16Phe-Asp[-OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Preparation (4d)
Operation according to 4a makes 6.59g (yield 99%) target compound with 7.58g (0.0098mol) 3d.ESI-MS(m/z)：673[M+H]
+.
Embodiment 17Boc-Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Preparation (5a)
In the 100ml eggplant-shape bottle, add 3.88g (0.012mol) Boc-Asp (OBzl), after anhydrous THF dissolving, in reaction solution, add 1.48g (0.011mol) HOBt and 2.26g (0.011mol) DCC, (the N end should be first with regulating between the pH8-9 with NMM after the anhydrous THF dissolving again to add 4.89 (0.0098mol) 4a behind the condition of ice bath stirring reaction 0.5h, add in the reaction solution again), after reaction is spent the night, TLC shows that N end raw material point disappears, filtering reacting liquid is removed DCU, again filtrate decompression is concentrated into dried, with 100ml acetic acid ethyl dissolution residue, filter and again remove insoluble DCU, ethyl acetate layer is used 5% sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, 5% aqueous potassium hydrogen sulfate, saturated sodium-chloride water solution, saturated sodium bicarbonate aqueous solution, each extraction of saturated sodium-chloride water solution is washed three times, tells the ethyl acetate layer anhydrous Na
2SO
4Dried overnight is filtered, and filtrate decompression is concentrated into dried, gets 7.85g (yield 99%) title compound, the off-white color solid.
(c＝1.2，CHCl
3)；ESI-MS(m/z)：833[M+Na]
+。
Embodiment 18Boc-Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Preparation (5b)
With 3.88g (0.012mol) Boc-Asp (OBzl), and 5.49g (0.0098mol) 4b makes 8.47g (yield 99%) title compound, off-white color solid according to the operation of 5a.
(c＝1，CHCl
3)；ESI-MS(m/z)：889[M+Na]
+。
Embodiment 19Boc-Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Preparation (5c)
Operation according to 5a makes 9.03g (yield 99%) title compound, off-white color solid with 3.88g (0.012mol) Boc-Asp (OBzl) and 6.04g (0.0098mol) 4c.Mp：70.0-71.5℃；
(c＝1，CHCl
3)；ESI-MS(m/z)：945[M+Na]
+。
Embodiment 20Boc-Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Preparation (5d)
Operation according to 5a makes 9.57g (yield 99%) title compound, white solid with 3.88g (0.012mol) Boc-Asp (OBzl) and 6.59g (0.098mol) 4d.Mp：80.5-81.9℃；
(c＝1，CHCl
3)；ESI-MS(m/z)：1001[M+Na]
+。
Embodiment 21Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Preparation (6a)
With a small amount of ethyl acetate 7.85 (0.0098mol) 5a is dissolved, ice bath stirs the lower 4N of adding HCl/ ethyl acetate solution to be mixed, and TLC shows that raw material point disappears, and the solution with water pump is drained repeatedly, the Ex-all hydrogen chloride gas gets 6.88g (yield 99%) target compound.ESI-MS(m/z)：711[M+H]
+.
Embodiment 22Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Preparation (6b)
Operation according to 5a makes 7.50g (yield 99%) target compound with 8.47g (0.0098mol) 5b.ESI-MS(m/z)：767[M+H]
+.
Embodiment 23Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Preparation (6c)
Operation according to 5a makes 8.05g (yield 99%) target compound with 9.03g (0.0098mol) 5c.ESI-MS(m/z)：823[M+H]
+.
Embodiment 24Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Preparation (6d)
Operation according to 5a makes 8.59g (yield 99%) target compound with 9.47g (0.0098mol) 5d.ESI-MS(m/z)：879[M+H]
+.
Embodiment 25Boc-Arg (N0
2The preparation of)-Gly-OMe
In the 100ml eggplant-shape bottle, add 3.19g (0.01mo) Boc-Arg (NO
2), with after the about anhydrous THF melt into of the 50ml homogeneous attitude, in reaction solution, add 1.48g (0.011mol) HOBt and 2.26g (0.011mol) DCC, add Gly-OMe 1.38g (0.011mol) behind the condition of ice bath stirring reaction 1h, regulate between the pH8-9 with NMM again, after reaction was spent the night, TLC showed Boc-Arg (NO
2) disappearance of raw material point, filtering reacting liquid is removed DCU, again filtrate decompression is concentrated into dried, with 100ml acetic acid ethyl dissolution residue, filter and again remove insoluble DCU, ethyl acetate layer is washed three times with 5% sodium bicarbonate aqueous solution, saturated sodium-chloride water solution, 5% aqueous potassium hydrogen sulfate, saturated sodium-chloride water solution, saturated sodium bicarbonate aqueous solution, each extraction of saturated sodium-chloride water solution successively, tells the ethyl acetate layer anhydrous Na
2SO
4Dried overnight is filtered, and filtrate decompression is concentrated into dried, gets 3.51g (yield 90%) title compound, light yellow oil.ESI-MS(m/z)：467[M+H]
+.
Embodiment 26Boc-Arg (NO
2The preparation of)-Gly
In the 100ml eggplant-shape bottle, with 20.0mlMeOH with 3.51g (0.009mol) Boc-Arg (NO
2)-Gly-OMe dissolves fully, add NaOH (2N) solution under the condition of ice bath, regulate pH12, keep condition of ice bath constant, TLC behind the 2h (chloroform/methanol/Glacial acetic acid, 5: 1: 0.1) shows the completely dissolve of raw material point, transfers pH to 7 with saturated aqueous potassium hydrogen sulfate, the concentrating under reduced pressure desolventizing adds 10mlH
2O, at ice bath agitation condition downward modulation pH to 2, solution is with ethyl acetate extraction three times (50ml * 3), and is extremely neutral with saturated NaCl washing ester layer again with saturated aqueous potassium hydrogen sulfate.Use anhydrous Na
2SO
4Dried overnight is filtered, and filtrate decompression is concentrated into dried, then separates with 200-300 order silica gel column chromatography, gets 2.37g (yield 70%) title compound, white solid.
(c＝1，CH
3OH)；ESI
--MS(m/z)：375[M-H]
+.
Embodiment 27
Boc-Arg (N0
2)-Gly-Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Preparation (7a)
In the 100ml eggplant-shape bottle, add 4.51g (0.012mol) Boc-Arg (NO
2)-Gly, after anhydrous THF dissolving, in reaction solution, add 1.62g (0.011mol) HOBt and 2.472g (0.0011mol) DCC, (the N end should be first with regulating between the pH8-9 with NMM after the anhydrous THF dissolving again to add 6a behind the condition of ice bath stirring reaction 0.5h, add in the reaction solution again), after reaction is spent the night, TLC shows that N end raw material point disappears, filtering reacting liquid is removed DCU, again filtrate decompression is concentrated into dried, with 100ml acetic acid ethyl dissolution residue, filter and again remove insoluble DCU, ethyl acetate layer is used 5% sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, 5% aqueous potassium hydrogen sulfate, saturated sodium-chloride water solution, saturated sodium bicarbonate aqueous solution, each extraction of saturated sodium-chloride water solution is washed three times, tells the ethyl acetate layer anhydrous Na
2SO
4Dried overnight is filtered, and filtrate decompression is concentrated into dried.Separate with 200-300 order silica gel column chromatography, get 5.44g (yield 50%) title compound, white solid.Mp：139.8-141.4℃；
(c＝1.7，CH
3OH)；ESI-MS(m/z)：1091[M+Na]
+。
Embodiment 28
Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Preparation (7b)
According to the operation of 7a with 4.51g (0.012mol) Boc-Arg (NO
2)-Gly, and 6b makes 5.61g (yield 50%) title compound, off-white color solid.Mp：146.6-149.3℃；
(c＝1，CH
3OH)；ESI-MS(m/z)：1147[M+Na]
+。
Embodiment 29
Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Preparation (7c)
According to the operation of 7a with 4.51g (0.012mol) Boc-Arg (NO
2After)-Gly and 6c make the target compound crude product, separate with 200-300 order silica gel column chromatography, get 5.30g (yield 45%) title compound, white solid.Mp：146.7-148.5℃；
(c＝1，CH
3OH)；ESI-MS(m/z)：1203[M+Na]
+。
Embodiment 30
Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-Asp[-OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Preparation (7d)
According to the operation of 7a with 4.51g (0.012mol) Boc-Arg (NO
2After)-Gly and 6d 8.59g (0.0098mol) make the target compound crude product, separate with 200-300 order silica gel column chromatography, get 5.56g (yield 45%) title compound, white solid.Mp：148.5-150.2℃；
(c＝1，CH
3OH)；ESI-MS(m/z)：1259[M+Na]
+。
Embodiment 31Boc-Arg-Gly-Asp-Phe-Asp[-OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Preparation (8a)
With 7a 0.20g dissolve with methanol, add Pd/C an amount of (10%), it is airtight that reaction system keeps, and passes into the repeatedly air in the replacement(metathesis)reaction system of hydrogen, keeps the hydrogen stirring at room.TLC detection reaction process behind the hydrogen is no longer inhaled in reaction.React complete after, normal pressure filter to be removed Pd/C, filtrate is ground to get title compound 0.17g (yield 95%) repeatedly with ether after being spin-dried for solvent.ESI-MS(m/z)：934[M+H]
+.
Embodiment 32Boc-Arg-Gly-Asp-Phe-Asp[-OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Preparation (8b)
Operation according to 8a makes title compound 0.17g (yield 95%) with 7b 0.20g.ESI-MS(m/z)：990[M+H]
+.
Embodiment 33Boc-Arg-Gly-Asp-Phe-Asp[-OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Preparation (8c)
Operation according to 8a makes title compound 0.17g (yield 95%) with 0.20g 7c.ESI-MS(m/z)：1046[M+H]
+.
Embodiment 34Boc-Arg-Gly-Asp-Phe-Asp[-OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Preparation (8d)
Operation according to 8a makes title compound 0.17g (yield 95%) with 0.20g 7d.ESI-MS(m/z)：1102[M+H]
+.
Embodiment 35Arg-Gly-Asp-Phe-Asp[-OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Preparation (9a)
With a small amount of ethyl acetate 0.17g (0.00017mol) 8a is dissolved, ice bath stirs the lower 4N of adding HCl/ ethyl acetate solution to be mixed, and TLC shows that raw material point disappears, and the solution with water pump is drained repeatedly, the Ex-all hydrogen chloride gas gets 0.15g (yield 99%) target compound.Mp：135.8-137.1；
(c＝1.4，CH
3OH)；ESI-MS(m/z)：834[M+H]
+。
Embodiment 36Arg-Gly-Asp-Phe-Asp[-OCH
2(CH
2)
8CH
3]]-OCH
2(CH
2)
8CH
3Preparation (9b)
Operation according to 9a makes 0.15g (yield 99%) target compound with 0.17g (0.00017mol) 8b.Mp：187.4-188.7℃；
(c＝1，CH
3OH)；ESI-MS(m/z)：890[M+H]
+。
Embodiment 37Arg-Gly-Asp-Phe-Asp[-OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Preparation (9c)
Operation according to 9a makes 0.15g (yield 99%) target compound with 0.17g (0.00016mol) 8c.Mp：189.9-192.0℃；
(c＝1，CH
3OH)；ESI-MS(m/z)：946[M+H]
+。
Embodiment 38Arg-Gly-Asp-Phe-Asp[-OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Preparation (9d)
Operation according to 9a makes 0.15g (yield 99%) target compound with 0.17g (0.00015mol) 8d.Mp：189.7-191.6℃；
(c＝1，CH
3OH)；ESI-MS(m/z)：1002[M+H]
+。
The antithrombotic acitivity test of test example 1 the compounds of this invention oral administration
1) rat operation and apparatus
(male, 190～210g) press 1nmolkg to the Wistar rat
-1Dosage is oral the compounds of this invention respectively, 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 respectively surgical thread, 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 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 for inserting 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; Then take out silk thread, 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 with syringe by sharp pipe end
-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: press respectively 1nmolkg
-1Dosage configuration the compounds of this invention 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 ophthalmic tweezers, dips in gently drop of blood on filter paper, puts in advance weighted penicillin bottle, accurately weighs and record.Calculate the weight in wet base of thrombus.Each medicine repeats 10 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 1 in.
Antithrombotic acitivity evaluation result in the oral body of table 1.
| Testing compound | Wet weight of thrombus (mg) |
| NS | 32.74±2.87 |
| Asprin | 14.42±2.90 a |
| 9a | 16.34±3.32 a |
| 9b | 19.33±3.94 a |
| 9c | 17.59±1.41 a |
| 9d | 16.23±2.04 a |
N=10; NS:0.9% physiological saline;
aCompare P＜0.01 with NS.
Test example 2 compounds of the present invention are in the diameter characterization test of aqueous phase self-assembly
4 kinds of conjugates of the present invention are according to 10
-3The concentration of mol/L is configured as the aqueous solution, nano particle size and Zeta potential determinator (Nano-ZS90, Britain Ma Erwen Zeta nano series) METHOD FOR CONTINUOUS DETERMINATION 8 days, observes change of size, the results are shown in Table 2.The data that obtain show, the particle diameter of these 4 kinds of compounds and eight days can both stable existence (as shown in Figure 2) all between 196 ± 40nm to 255 ± 78nm.Thereby be outstanding micro emulsion and the preparation material of liposome medicament.
Table 2. the compounds of this invention particle diameter and Zeta
| Compound | The 1st day | The 2nd day | The 3rd day | The 4th day | The 5th day | The 6th day | The 7th day | The 8th day | Particle diameter (nm) | Zeta (mv) |
| 9a | 299 | 387 | 201 | 207 | 178 | 225 | 202 | 346 | 255±78 | -135 |
| 9b | 371 | 290 | 151 | 178 | 163 | 141 | 314 | 242 | 231±86 | 75.5 |
| 9c | 286 | 210 | 195 | 168 | 200 | 155 | 182 | 174 | 196±40 | -2.31 |
| 9d | 286 | 275 | 199 | 243 | 243 | 172 | 285 | 232 | 241±40 | -11.3 |
Test example 3 compounds of the present invention characterize at the TEM of aqueous phase self-assembly
According to the mensuration requirement of TEM, compound is dissolved in to be mixed with concentration in the distilled water be 10
-3The solution of mol/L drops on the special 200 order copper mesh, and room temperature volatilizes naturally, at transmission electron microscope (TEM) its form of lower observation and particle diameter, and uses the photo record.The result can be along with the increase of aliphatic chain chain length, and compound may have the trend of gathering.9a, 9b, 9c, 9d be along with the increase of aliphatic chain carbon number, that TEM result begins to become is intensive (Fig. 3-Fig. 6).
Test example 4 compounds of the present invention characterize at the SEM of aqueous phase self-assembly
According to the mensuration requirement of SEM, compound is dissolved in to be mixed with concentration in the distilled water be 10
-3The solution of mol/L is lyophilized into powder.In its form of the lower observation of scanning electronic microscope (SEM), and use the photo record.By picture as can be known from 9a to 9d along with the increase of carbochain, fibre pipe is netted and links to each other.Along with the growth of carbochain, it is intensive that nanostructure begins to become, and aggregation tendency (Fig. 7-Figure 12) may be arranged.
Claims (5)
1. the conjugate of a class peptide chain and double fatty alcohol chain is the conjugate of Arg-Gly-Asp-Phe-Asp peptide chain and two aliphatic alcohol chains, and the general formula of described conjugate is
Arg-Gly-Asp-Phe-Asp[-OCH
2(CH
2)
nCH
3]-OCH
2(CH
2)
nCH
3, n=6 wherein, 8,10 or 12.
2. method for preparing the conjugate of peptide chain claimed in claim 1 and double fatty alcohol chain, its step comprises:
Aspartic acid and two Fatty Alcohol(C12-C14 and C12-C18) condensations of (1) N being held protecting group protect, described Fatty Alcohol(C12-C14 and C12-C18) is CH
3(CH
2)
nCH
2OH, n=6,8,10,12;
(2) slough N end protecting group and obtain the double fatty alcohol aspartate;
(3) according to existing liquid phase synthetic technology, the double fatty alcohol aspartate is progressively connect peptide chain and the double fatty alcohol chain conjugate that the synthetic protecting group of peptide is protected with the protection intermediate of phenylalanine, the protection intermediate of aspartic acid, protection intermediate and the arginic protection intermediate of glycine successively;
(4) slough successively the peptide chain of protecting group protection and C end protecting group and the N end protecting group of double fatty alcohol chain conjugate and obtain target compound.
3. the preparation method of the conjugate of peptide chain according to claim 2 and double fatty alcohol chain the steps include:
(1) in the presence of DCC, HOBt, NMM and THF with the aspartic acid of tertbutyloxycarbonyl protection respectively with CH
3(CH
2)
7OH, CH
3(CH
2)
9OH, CH
3(CH
2)
11OH and CH
3(CH
2)
13The OH coupling;
(2) in hydrogenchloride/ethyl acetate solution, add the conjugate of step (1) gained, slough tertbutyloxycarbonyl;
(3) phenylalanine and step (2) the products therefrom coupling in the presence of DCC, HOBt, NMM and THF, tertbutyloxycarbonyl protected;
(4) in hydrogenchloride/ethyl acetate solution, add the conjugate of step (3) gained, slough tertbutyloxycarbonyl;
Aspartic acid and step (4) the products therefrom coupling of (5) in the presence of DCC, HOBt, NMM and THF, holding tertbutyloxycarbonyl protection, side chain benzyloxy to protect N;
(6) in hydrogenchloride/ethyl acetate solution, add the conjugate of step (5) gained, slough tertbutyloxycarbonyl;
The glycine coupling of (7) in the presence of DCC, HOBt, NMM and THF, N being held arginine and the methoxyl group of tertbutyloxycarbonyl protection, the protection of side chain nitro to protect;
(8) in the presence of 2NNaOH, (7) products therefrom is removed C and hold protecting group;
(9) in the presence of DCC, HOBt, NMM and THF with (6) and (8) products therefrom coupling;
(10) (9) products therefrom deprotection base is obtained compound shown in the general formula.
4. the conjugate of peptide chain claimed in claim 1 and double fatty alcohol chain is in the purposes of preparation in the antithrombotic reagent.
5. the conjugate of peptide chain claimed in claim 1 and double fatty alcohol chain is in the purposes of preparation micro emulsion or liposome medicament.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1099760A (en) * | 1993-04-01 | 1995-03-08 | 默克专利股份有限公司 | Cyclic adhesion inhibitors |
| CN101240026A (en) * | 2007-02-07 | 2008-08-13 | 首都医科大学 | Conjugate constructed from normal tridecyl and RGD peptide, and its synthesis and application in medicine |
| CN101401941A (en) * | 2008-11-21 | 2009-04-08 | 首都医科大学 | Preparation and uses of tumor-targeted carrier material RGD-fatty alcohol series of compounds |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1099760A (en) * | 1993-04-01 | 1995-03-08 | 默克专利股份有限公司 | Cyclic adhesion inhibitors |
| CN101240026A (en) * | 2007-02-07 | 2008-08-13 | 首都医科大学 | Conjugate constructed from normal tridecyl and RGD peptide, and its synthesis and application in medicine |
| CN101401941A (en) * | 2008-11-21 | 2009-04-08 | 首都医科大学 | Preparation and uses of tumor-targeted carrier material RGD-fatty alcohol series of compounds |
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