CN102477071B - Pro-Ala-Lys-Lys(Pro-Ala-Lys)-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3, synthesis method and application thereof as thrombus dissolving agent - Google Patents
Pro-Ala-Lys-Lys(Pro-Ala-Lys)-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3, synthesis method and application thereof as thrombus dissolving agent Download PDFInfo
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- CN102477071B CN102477071B CN 201010573551 CN201010573551A CN102477071B CN 102477071 B CN102477071 B CN 102477071B CN 201010573551 CN201010573551 CN 201010573551 CN 201010573551 A CN201010573551 A CN 201010573551A CN 102477071 B CN102477071 B CN 102477071B
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- 208000007536 Thrombosis Diseases 0.000 title claims abstract description 56
- FYQSMXKJYTZYRP-DCAQKATOSA-N Pro-Ala-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 FYQSMXKJYTZYRP-DCAQKATOSA-N 0.000 title 1
- 238000001308 synthesis method Methods 0.000 title 1
- 238000002360 preparation method Methods 0.000 claims abstract description 44
- 230000000694 effects Effects 0.000 claims abstract description 31
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- 238000000034 method Methods 0.000 claims description 47
- CIOWSLJGLSUOME-BQBZGAKWSA-N Lys-Asp Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC(O)=O CIOWSLJGLSUOME-BQBZGAKWSA-N 0.000 claims description 20
- 238000009833 condensation Methods 0.000 claims description 20
- 108010003700 lysyl aspartic acid Proteins 0.000 claims description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- -1 dicyclohexyl carbonyl diimine Chemical compound 0.000 claims description 13
- 230000005494 condensation Effects 0.000 claims description 12
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 11
- 238000007327 hydrogenolysis reaction Methods 0.000 claims description 8
- MVEAAGBEUOMFRX-UHFFFAOYSA-N ethyl acetate;hydrochloride Chemical compound Cl.CCOC(C)=O MVEAAGBEUOMFRX-UHFFFAOYSA-N 0.000 claims description 7
- 239000003527 fibrinolytic agent Substances 0.000 claims description 6
- 229960000103 thrombolytic agent Drugs 0.000 claims description 6
- 229920006395 saturated elastomer Polymers 0.000 claims description 5
- 150000002191 fatty alcohols Chemical class 0.000 claims description 4
- 229910000071 diazene Inorganic materials 0.000 claims description 3
- 150000003851 azoles Chemical class 0.000 claims 1
- 229910052757 nitrogen Inorganic materials 0.000 claims 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen(.) Chemical compound [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract 1
- 229940079593 drug Drugs 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 description 60
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 40
- 239000000843 powder Substances 0.000 description 40
- 239000000243 solution Substances 0.000 description 25
- 230000002537 thrombolytic effect Effects 0.000 description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 13
- 239000002504 physiological saline solution Substances 0.000 description 11
- 239000004698 Polyethylene Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 229920000573 polyethylene Polymers 0.000 description 10
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Substances C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 10
- 238000005481 NMR spectroscopy Methods 0.000 description 9
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- 229920001971 elastomer Polymers 0.000 description 7
- 229910001220 stainless steel Inorganic materials 0.000 description 7
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 6
- 208000020442 loss of weight Diseases 0.000 description 6
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- 102000004196 processed proteins & peptides Human genes 0.000 description 6
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
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- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 3
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
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- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 2
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- JGSARLDLIJGVTE-UHFFFAOYSA-N 3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a thrombus dissolving oligopeptide and a preparation method and application thereof. The oligopeptide has thrombus dissolving activities and has a structural formula shown in the general formula I in the specification, wherein in the formula, n is 6, 8, 10, 12, 14 or 16. The thrombus dissolving activity experiments indicate that the oligopeptide shown in the general formulaI has excellent thrombus dissolving activities and can be applied as a thrombus dissolving drug.
Description
Technical field
The present invention relates to oligopeptides, relate in particular to oligopeptides and synthetic method thereof with thrombus dissolving activity, the invention still further relates to them as the application of thrombolytic agent, the invention belongs to the oligopeptides field.
Background technology
P6A (ARPAK) is one of scleroproein β chain degradation product, has thrombus dissolving activity.In the metabolism research of P6A, found meta-bolites PAK.On rat arteriovenous shut intubate thrombus dissolving model, the thrombus dissolving activity of PAK is stronger than parent P6A.According to general understanding, polypeptide all can be degraded rapidly in vivo.Structural modification by PAK delays vivo degradation speed and improves thrombus dissolving activity, is the important channel of oligopeptides thrombolytic agent research.
According to general understanding, contain the amphipathic molecule of polypeptide, for example self-assembly can take place by intermolecular non-covalent interaction in aliphatic alcohol chain modified polypeptides under suitable condition, forms nanostructure.By nanostructure can improve in vivo conveying of polypeptide, delay polypeptide in vivo degradation rate and improve the activity in vivo of polypeptide.According to these understanding, the contriver has proposed the present invention.
Summary of the invention
One of the object of the invention provides the oligopeptides with thrombus dissolving activity;
Two of the object of the invention provides a kind of synthetic above-mentioned method with thrombus dissolving activity oligopeptides.
Three of the object of the invention is that above-mentioned oligopeptides is applied to prepare thrombolytic agent.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Oligopeptides with thrombus dissolving activity, its structural formula are shown in the general formula I:
N=6,8,10,12,14 or 16 in the formula.
A kind of method of the synthetic described oligopeptides of above-mentioned general formula I may further comprise the steps:
1), Boc-Pro and Ala-OBzl condensation are Boc-Pro-Ala-OBzl;
2) be Boc-Pro-Ala with the Boc-Pro-Ala-OBzl hydrogenolysis;
3), Boc-Pro-Ala and Lys (Boc)-OBzl condensation is Boc-Pro-Ala-Lys (Boc)-OBzl;
4) be Boc-Pro-Ala-Lys (Boc) with Boc-Pro-Ala-Lys (Boc)-OBzl hydrogenolysis;
5), Boc-Asp and saturated fatty alcohol condensation are Boc-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3, n=6,8,10,12,14 or 16 wherein;
6), Boc-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Slough Boc and generate Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3
7), Boc-Lys (Boc) and Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Condensation is Boc-Lys (Boc)-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3
8), Boc-Lys (Boc)-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Remove Boc and generate Lys-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3
9), Boc-Pro-Ala-Lys (Boc) and Lys-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Condensation is Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2(CH
2) n-CH
3]-OCH
2(CH
2) nCH
3
10), Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2-(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Remove Boc.
In order to reach better synthetic effect, step 1) is Boc-Pro-Ala-OBzl with the Ala-OBzl condensation at Boc-Pro in the presence of the DCCHOBt in anhydrous THF preferably; Step 2) in methyl alcohol, be Boc-Pro-Ala with the Boc-Pro-Ala-OBzl hydrogenolysis preferably; Step 3) preferably in the presence of DCC and HOBt Boc-Pro-Ala in anhydrous THF, be Boc-Pro-Ala-Lys (Boc)-OBzl with Lys (Boc)-OBzl condensation;
Step 4) is Boc-Pro-Ala-Lys (Boc) with Boc-Pro-Ala-Lys (Boc)-OBzl hydrogenolysis in methyl alcohol preferably; Step 5) is Boc-Asp[OCH with the saturated fatty alcohol condensation at Boc-Asp in the presence of the DCC in anhydrous THF preferably
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Step 6) is Boc-Asp[OCH in hydrogenchloride-ethyl acetate solution preferably
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Take off Boc and generate Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Step 7) preferably in the presence of DCC and HOBt Boc-Lys (Boc) in anhydrous THF with Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Condensation is Boc-Lys (Boc)-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Step 8) is Boc-Lys (Boc)-Asp[OCH in hydrogenchloride-ethyl acetate solution preferably
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Remove Boc and generate Lys-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Step 9) preferably in the presence of DCC and HOBt Boc-Pro-Ala-Lys (Boc) in anhydrous THF with Lys-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Condensation is Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Step 10) is Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc) in hydrogenchloride-ethyl acetate solution preferably]-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Take off Boc.
The interior thrombus dissolving activity test of external and body shows that the oligopeptide compounds shown in the general formula I of the present invention has outstanding thrombus dissolving activity activity, can be used as thrombolytic agent and uses.
The breviary term
The THF tetrahydrofuran (THF)
HOBT N-hydroxy benzo triazole
DCC dicyclohexyl carbonyl diimine
The TLC thin-layer chromatography
The Boc tertbutyloxycarbonyl
Description of drawings
The structural formula of Fig. 1 compound of Formula I of the present invention.
The synthetic route chart of Fig. 2 compound of Formula I of the present invention; I) DCC, HOBt and NMM; Ii) Pd/C, H
2Iii) HCl/EA.
The transmission electron microscope photo of Fig. 3 The compounds of this invention 6b.
Embodiment
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 1 preparation Boc-Asp[OCH
2(CH
2)
6CH]-OCH
2(CH
2)
6CH
3(1a)
3.0g (12.9mmol) Boc-Asp is dissolved with the anhydrous THF of 20ml.In the solution that obtains, add 4.17g (30.9mmol) N-hydroxy benzo triazole (HOBT).After 10 minutes, under ice bath, add the solution of 6.37g (30.9mmol) dicyclohexyl carbonyl diimine (DCC) and the anhydrous THF of 25ml, obtain reaction solution (I).4.02g (30.9mmol) aliphatic alcohol chain is dissolved in the anhydrous THF of 10ml and stirred 30 minutes, obtain reaction solution (II).Under the ice bath reaction solution (II) is added in the reaction solution (I), stirring at room 12h then, (petrol ether/ethyl acetate 15:1) shows that Boc-Asp disappears to TLC.Reaction mixture filters, filtering dicyclohexylurea (DCU) (DCU).Filtrate decompression concentrates, and removes THF.Residue 250ml acetic acid ethyl dissolution.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.Ethyl acetate is used anhydrous Na mutually
2SO
4Drying, filtration, filtrate decompression are concentrated into dried, and residue obtains 4.23g (72%) target compound through column chromatography purification, is colourless powder.ESI-MS(m/e):458[M+H]
+.
Embodiment 2 preparation Boc-Asp[OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3(1b)
According to the method for embodiment 1 by 5.0g (21.5mmol) Boc-Asp and 8.14g (51.5mmol) CH
3(CH
2)
8CH
2OH makes 7.60g (69%) target compound, is colourless powder.ESI-MS(m/e):514[M+H]
+.
According to the method for embodiment 1 by 5.0g (21.5mmol) Boc-Asp and 9.58g (51.5mmol) CH
3(CH
2)
10CH
2OH makes 7.94g (65%) target compound, is colourless powder.ESI-MS(m/e):570[M+H]
+.
Embodiment 4 preparation Boc-Asp[OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3(1d)
According to the method for embodiment 1 by 5.0g (21.5mmol) Boc-Asp and 11.02g (51.5mmol) CH
3(CH
2)
12CH
2OH makes 8.32g (62%) target compound, is colourless powder.ESI-MS(m/e):626[M+H]
+.
Embodiment 5 preparation Boc-Asp[OCH
2(CH
2)
14CH
3]-OCH
2(CH
2)
14CH
3(1e)
According to the method for embodiment 1 by 3.0g (12.9mmol) Boc-Asp and 7.48g (30.9mmol) CH
3(CH
2)
14CH
2OH makes 5.16g (59%) target compound, is colourless powder.ESI-MS(m/e):682[M+H]
+.
Embodiment 6 preparation Boc-Asp[OCH
2(CH
2)
16CH
3]-OCH
2(CH
2)
16CH
3(1f)
According to the method for embodiment 1 by 3.0g (12.9mmol) Boc-Asp and 8.34g (30.9mmol) CH
3(CH
2)
16CH
2OH makes 4.94g (52%) target compound, is colourless powder.ESI-MS(m/e):738[M+H]
+.
Embodiment 7 preparation Asp[OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3(2a)
With 4.23g (9.26mmol) Boc-Asp[OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Be dissolved in 45ml 4N hydrogenchloride-ethyl acetate solution, stirring at room 1 hour, TLC (petrol ether/ethyl acetate, 15: 1) shows Boc-Asp (OC
8H
17)-OC
8H
17Disappear, 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 de-chlorine hydride.Add a small amount of ether at last residue is ground to form 3.39g (93%) target compound, be colourless powder.ESI-MS(m/e):358[M+H]
+.
Embodiment 8 preparation Asp[OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3(2b)
Method 5.96g (11.6mmol) Boc-Asp[OCH according to embodiment 7
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Make 4.96g (95%) target compound, be colourless powder.ESI-MS(m/e):414[M+H]
+.
Embodiment 9 preparation Asp[OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3(2c)
Method 6.28g (11.0mmol) Boc-Asp[OCH according to embodiment 7
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Make 5.24g (94%) target compound, be colourless powder.ESI-MS(m/e):470[M+H]
+.
Embodiment 10 preparation Asp[OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3(2d)
Method 6.32g (10.1mmol) Boc-Asp[OCH according to embodiment 7
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Make 5.39g (95%) target compound, be colourless powder.ESI-MS(m/e):526[M+H]
+.
Embodiment 11 preparation Asp[OCH
2(CH
2)
14CH
3]-OCH
2(CH
2)
14CH
3(2e)
Method 5.18g (7.6mmol) Boc-Asp[OCH according to embodiment 7
2(CH
2)
14CH
3]-OCH
2(CH
2)
14CH
3Make 4.27g (91%) target compound, be colourless powder.ESI-MS(m/e):582[M+H]
+.
Embodiment 12 preparation Asp[OCH
2(CH
2)
16CH
3]-OCH
2(CH
2)
16CH
3(2f)
Method 4.94g (6.7mmol) Boc-Asp[OCH according to embodiment 7
2(CH
2)
16CH
3]-OCH
2(CH
2)
16CH
3Make 4.06g (90%) target compound, be colourless powder.ESI-MS(m/e):638[M+H]
+.
Embodiment 13 preparation Boc-Lys (Boc)-Asp[OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3(3a)
According to the method for embodiment 1 by 1.25g (3.6mmol) Boc-Lys (Boc) and 1.18g (3.0mmol) Asp[OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Make 1.63g (80%) target compound, be colourless powder.ESI-MS(m/e):686[M+H]
+.
Embodiment 14 preparation Boc-Lys (Boc)-Asp[OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3(3b)
According to the method for embodiment 1 by 1.25g (3.6mmol) Boc-Lys (Boc) and 1.35g (3.0mmol) Asp[OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Make 1.68g (75%) target compound, be colourless powder.ESI-MS(m/e):742[M+H]
+.
Embodiment 15 preparation Boc-Lys (Boc)-Asp[OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3(3c)
According to the method for embodiment 1 by 1.25g (3.6mmol) Boc-Lys (Boc) and 1.52g (3.0mmol) Asp[OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Make 1.72g (72%) target compound, be colourless powder.ESI-MS(m/e):798[M+H]
+.
Embodiment 16 preparation Boc-Lys (Boc)-Asp[OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3(3d)
According to the method for embodiment 1 by 1.25g (3.6mmol) Boc-Lys (Boc) and 1.69g (3.0mmol) Asp[OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Make 1.72g (67%) target compound, be colourless powder.ESI-MS(m/e):854[M+H]
+.
Embodiment 17 preparation Boc-Lys (Boc)-Asp[OCH
2(CH
2)
14CH
3]-OCH
2(CH
2)
14CH
3(3e)
According to the method for embodiment 1 by 1.25g (3.6mmol) Boc-Lys (Boc) and 1.85g (3.0mmol) Asp[OCH
2(CH
2)
14CH
3]-OCH
2(CH
2)
14CH
3Make 1.77g (65%) target compound, be colourless powder.ESI-MS(m/e):910[M+H]
+.
Embodiment 18 preparation Boc-Lys (Boc)-Asp[OCH
2(CH
2)
16CH
3]-OCH
2(CH
2)
16CH
3(3f)
According to the method for embodiment 1 by 0.83g (2.4mmol) Boc-Lys (Boc) and 1.35g (2.0mmol) Asp[OCH
2(CH
2)
16CH
3]-OCH
2(CH
2)
16CH
3Make 1.64g (85%) target compound, be colourless powder.ESI-MS(m/e):966[M+H]
+.
Embodiment 19 preparation Lys-Asp[OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3(4a)
According to the method for embodiment 7 by 1.63g (2.4mmol) Boc-Lys (Boc)-Asp[OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3Make 1.17g (88%) target compound, be colourless powder.ESI-MS(m/e):486[M+H]
+.
Embodiment 20 preparation Lys-Asp[OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3(4b)
According to the method for embodiment 7 by 1.68g (2.26mmol) Boc-Lys (Boc)-Asp[OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Make 1.27g (91%) target compound, be colourless powder.ESI-MS(m/e):542[M+H]
+.
Embodiment 21 preparation Lys-Asp[OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3(4c)
According to the method for embodiment 7 by 1.72g (2.16mmol) Boc-Lys (Boc)-Asp[OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Make 1.34g (93%) target compound, be colourless powder.ESI-MS(m/e):598[M+H]
+.
Embodiment 22 preparation Lys-Asp[OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3(4d)
According to the method for embodiment 7 by 1.72g (2.02mmol) Boc-Lys (Boc)-Asp[OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Make 1.301g (89%) target compound, be colourless powder.ESI-MS(m/e):654[M+H]
+.
Embodiment 23 preparation Lys-Asp[OCH
2(CH
2)
14CH
3]-OCH
2(CH
2)
14CH
3(4e)
According to the method for embodiment 7 by 1.77g (1.95mmol) Boc-Lys (Boc)-Asp[OCH
2(CH
2)
14CH
3]-OCH
2(CH
2)
14CH
3Make 1.39g (91%) target compound, be colourless powder.ESI-MS(m/e):710[M+H]
+.
Embodiment 24 preparation Lys-Asp[OCH
2(CH
2)
16CH
3]-OCH
2(CH
2)
16CH
3(4f)
According to the method for embodiment 7 by 1.64g (1.70mmol) Boc-Lys (Boc)-Asp[OCH
2(CH
2)
16CH
3]-OCH
2(CH
2)
16CH
3Make 1.35g (95%) target compound, be colourless powder.ESI-MS(m/e):766[M+H]
+.
Embodiment 25 preparation Boc-Pro-Ala-OBzl
Method according to embodiment 1 makes 5.19g (99%) target compound from 3.0g (14.0mmol) Boc-Pro and 5.88g (16.7mmol) TosAla-Obzl, is yellow powder.ESI-MS(m/e):377[M+H]
+.
Embodiment 26 preparation Boc-Pro-Ala
With 5.19g (13.8mmol) Boc-Pro-Ala-OBzl 25ml dissolve with methanol.In solution, add 1.0g Pd/C, logical H
2(0.02Mba) and stirring at room disappear to Boc-Pro-Ala-OBzl.Filtering Pd/C, filtrate decompression are concentrated into dried.Make 3.59g (91%) target compound, be yellow powder.ESI-MS(m/e):285[M-H]
-.
Embodiment 27 preparation Boc-Pro-Ala-Lys (Boc)-OBzl
Method according to embodiment 1 makes 6.78g (57%) target compound by 6.76g (23.6mmol) Boc-Pro-Ala and 10g (19.7mmol) TosLys (Boc)-OBzl, is colourless powder.ESI-MS(m/e):605[M+H]
+.
Embodiment 28 preparation Boc-Pro-Ala-Lys (Boc)
Method according to embodiment 14 makes 5.36g (93%) target compound by 6.78g (11.2mmol) Boc-Pro-Ala-Lys (Boc)-OBzl, is colourless powder.ESI-MS(m/e):513[M-H]
-.
Embodiment 29 preparation Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2(CH
2)
6CH
3]-OCH
2(CH
2)
6CH
3(5a)
According to the method for embodiment 1 by 1.32g (2.56mmol) Boc-Pro-Ala-Lys (Boc) and 650mg (1.16mmol) Lys-Asp (OC
8H
17)-OC
8H
17Make 180mg (11%) target compound, be colourless powder.Mp?163℃;[α]
D 25=-648(c=0.51,CH
3OH);ESI-MS(m/e):1478[M+H]
+.IR(KBr):3294,3235,2974,2932,2860,1639,1516,1456,1398,1393,1366,1273,1250,1223,1171,1121.
1H?NMR(500MHz,CDCl
3):δ/ppm=7.71-7.40(m,3H),7.23-7.21(m,1H),7.02-6.93(m,2H),5.22-4.96(m,2H),4.83-4.81(m,1H),4.54-4.49(m,1H),4.42-4.28(m,4H),4.24-4.16(m,2H),4.11-4.04(m,4H),3.47-3.39(m,4H),3.26-3.16(m,1H),3.07(s,4H),2.94-2.81(m,2H),2.16-2.02(m,4H),1.94-1.82(m,7H),1.73-1.67(m,3H),1.62-1.58(m,5H),1.46-1.41(m,41H),1.36-1.26(m,33H),0.88-0.86(t,J=5.5Hz,J=7.0Hz,6H).
Embodiment 30 preparation Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3(5b)
According to the method for embodiment 1 by 1.20g (2.33mmol) Boc-Pro-Ala-Lys (Boc) and 650mg (1.06mmol) Lys-Asp (OC
10H
21)-OC
10H
21Make 185mg (11%) target compound, be colourless powder.Mp?130℃;[α]
D 25=-568(c=0.59,CH
3OH);ESI-MS(m/e):1556[M+Na]
+;IR(KBr):3401,3283,3237,3208,3088,2976,2932,2857,2359,2332,1738,1634,1545,1516,1506,1479,1456,1393,1366,1277,1250,1229,1207,1167,1123,775.?
1H?NMR(500MHz,CDCl
3):δ/ppm=7.49-7.32(m,2H),7.26-7.07(m,1H),6.99-6.88(m,2H),5.21-5.07(m,2H),4.85-4.82(m,1H),4.52-4.35(m,5H),4.26-4.21(m,2H),4.14-4.06(m,4H),3.72-3.68(m,1H),3.56-3.48(m,5H),3.31-3.21(m,1H),3.14-3.08(m,4H),2.98-2.81(m,2H),2.19-1.93(m,14H),1.74-1.71(m,2H),1.62-1.59(m,5H),1.52-1.39(m,50H),1.33-1.28(m,30H),0.91-?0.88(t,J=6.5Hz,J=7.0Hz,6H).
Embodiment 31 preparation Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3(5c)
According to the method for embodiment 1 by 1.18g (2.30mmol) Boc-Pro-Ala-Lys (Boc) and 700mg (1.04mmol) Lys-Asp (OC
12H
25)-OC
12H
25Make 210mg (13%) target compound, be colourless powder.Mp?156℃;[α]
D 25=-467(c=0.72,CH
3OH);ESI-MS(m/e):1612[M+Na]
+;IR(KBr):3316,3296,3285,3252,3244,3046,2978,2926,2855,1738,1690,1639,1533,1516,1506,1476,1456,1395,1366,1277,1252,1169,1123,984,779.
1H?NMR(300MHz,CDCl
3):δ/ppm=7.88-7.71(m,1H),7.47-7.37(m,2H),7.21-7.11(m,1H),6.94-6.85(m,2H),5.08-4.98(m,1H),4.84-4.81(m,1H),4.46-4.36(m,4H),4.22(s,2H),4.12-3.99(m,4H),3.48-3.46(m,4H),3.22(s,2H),3.08-3.07(m,4H),2.93-2.81(m,2H),2.13-2.02(m,3H),1.92-1.89(m,6H),1.76-1.58(m,7H),1.46-1.37(m,43H),1.32-1.22(m,38H),0.89-0.86(t,J=5.7Hz,J=6.9Hz,6H).
Embodiment 32 preparation Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3(5d)
According to the method for embodiment 1 by 1.17g (2.27mmol) Boc-Pro-Ala-Lys (Boc) and 750mg (1.03mmol) Lys-Asp (OC
14H
29)-OC
14H
29Make 313mg (18%) target compound, be colourless powder.Mp?135℃;[α]
D 25=-946(c=0.34,CH
3OH);ESI-MS(m/e):1646[M+H]
+;IR(KBr):3292,2976,2926,2857,1738,1699,1686,1639,1530,1506,1456,1393,1366,1277,1450,1207,1173,1119.
1H?NMR(500MHz,CDCl
3):δ/ppm=7.66-7.39(m,2H),7.22-7.13(m,1H),7.01-6.91(m,2H),5.23-4.96(m,2H),4.83-4.81(m,1H),4.53-4.34(m,5H),4.22(s,2H),4.11-4.04(m,4H),3.51-3.43(m,4H),3.24-3.17(m,1H),3.08(s,4H),2.95-2.84(m,2H),2.50(s,1H),2.15-2.03(m,4H),1.94-1.83(m,7H),1.72-1.69(m,2H),1.62-1.58(m,5H),1.46-1.42(m,42H),1.37-1.25(m,58H),0.88-0.86(t,J=6.5Hz,J=6.5Hz,6H).
Embodiment 33 preparation Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2(CH
2)
14CH
3]-OCH
2(CH
2)
14CH
3(5e)
According to the method for embodiment 1 by 1.16g (2.25mmol) Boc-Pro-Ala-Lys (Boc) and 800mg (1.02mmol) Lys-Asp (OC
16H
33)-OC
16H
33, make 326mg (19%) target compound through column chromatography, be colourless powder.Mp?127℃;[α]
D 25=-865(c=0.37,CH
3OH);ESI-MS(m/e):1724[M+Na]
+;IR(KBr):3289,2926,2855,1738,1734,1690,1639,1516,1506,1447,1395,1366,1275,1252,1207,1173,1121.
1H?NMR(500MHz,CDCl
3):δ/ppm=7.45-7.32(m,2H),7.20-7.09(m,1H),6.91-6.81(m,2H),5.09-4.95(m,1H),4.85-4.81(m,1H),4.53-4.34(m,5H),4.22(s,2H),4.11-4.01(m,4H),3.72-3.68(m,2H),3.55-3.47(m,4H),3.29-3.23(m,1H),3.08(s,4H),3.01-2.77(m,2H),2.39-2.28(m,2H),2.21-2.02(m,4H),1.96-1.82(m,7H),1.72-1.67(m,3H),1.61-1.55(m,5H),1.47-1.42(m,40H),1.38-1.35(m,10H),1.28-1.19(m,54H),0.89-0.87(t,J=6.0Hz,J=7.0Hz,6H).
Embodiment 34 preparation Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2(CH
2)
16CH
3]-OCH
2(CH
2)
16CH
3(5f)
According to the method for embodiment 1 by 1.15g (2.23mmol) Boc-Pro-Ala-Lys (Boc) and 850mg (1.01mmol) Lys-Asp (OC
18H
37)-OC
18H
37Make 241mg (14%) target compound, be colourless powder.Mp?138℃;[α]
D 25=-905(c=0.35,CH
3OH);ESI-MS(m/e):1759[M+H]
+;IR(KBr):3298,3286,2926,2854,1687,1639,1529,1456,1392,1365,1274,1246,1170,1118.
1H-NMR(500MHz,CDCl
3):δ/ppm=7.43-7.36(m,1H),7.19(s,1H),6.919-6.88(m,2H),5.06-4.83(m,2H),4.52-4.35(m,5H),4.23(s,2H),4.13-4.06(m,5H),3.72-3.68(m,1H),3.51-3.48(m,5H),3.25(s,1H),3.09(s,4H),2.92-2.83(m,2H),2.22-2.08(m,5H),1.94-1.89(m,8H),1.73-1.69(m,5H),1.63-1.59(m,6H),1.48-1.42(m,37H),1.39-1.34(m,9H),1.33-1.27(m,64H),0.90-0.88(t,J=6.0Hz,J=7.0Hz,6H).
Embodiment 35 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[OCH
2(CH
2)
6CH
3]-OCH
2-(CH
2)
6CH
3(6a)
According to the method for embodiment 7 by 135mg (0.09mmol) Boc-Pro-Ala-Lys (Boc)-Lys (Boc-Pro-Ala-Lys)-Asp[OCH
2(CH
2)
8CH
3]-OCH
2(CH
2)
8CH
3Make 106mg (95%) target compound, be colourless powder.Mp?132℃;[α]
D 25=-134(c=0.51,H
2O);ESI-MS(m/e):1078[M+H]
+;IR(KBr):3437,3387,3250,3242,3227,3157,3078,3057,2928,2857,1734,1653,1616,1533,1456,1362,1287,1240,1206,1171.
1H?NMR(500MHz,DMSO-d
6):δ/ppm=10.05(s,1H),8.85-8.84(d,J=7.0Hz,1H),8.57-8.44(m,1H),8.18-8.07(m,3H),4.64-4.62(m,1H),4.36-4.34(m,2H),4.26-4.18(m,5H),4.03-4.00(m,4H),3.23-3.19(m,4H),3.05-2.97(m,2H),2.74-2.71(m,6H),2.37-2.30(m,2H),1.89-1.83(m,6H),1.69-1.63(m,3H),1.58-1.52(m,11H),1.38-1.22(m,36H),0.87-0.85(t,J=5.5Hz,J=7.0Hz,6H).
Embodiment 36 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[OCH
2(CH
2)
8CH
3]-OCH
2-(CH
2)
8CH
3(6b)
According to the method for embodiment 7 by 100mg (0.07mmol) Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[OCH
2(CH
2)
10CH
3]-OCH
2-(CH
2)
10CH
3Make 76mg (91%) target compound, be colourless powder.Mp?134℃;[α]
D 25=-78(c=0.59,H
2O);ESI-MS(m/e):1134[M+H]
+;IR(KBr):3437,3420,3404,3377,3356,3264,3248,3057,3021,2955,2928,2857,2762,2656,1740,1657,1547,1458,1391,1288,1240,1225,1207,1171,727.
1H-NMR(500MHz,DMSO-d
6):δ/ppm=10.09(s,1H),8.86(s,1H),8.44-8.43(d,J=7.5Hz,1H),8.34-7.96(m,5H),4.64-4.62(d,J=5.5Hz,1H),4.36-4.32(m,2H),4.23-4.18(m,5H),4.06-3.96(m,4H),3.26-3.18(m,4H),3.04-2.97(m,2H),2.75-2.73(m,6H),2.37-2.31(m,2H),1.91-1.81(m,6H),1.68-1.54(m,14H),1.36-1.22(m,43H),0.86-0.84(m,6H).
Embodiment 35 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3(6c)
According to the method for embodiment 7 by 110mg (0.07mmol) Boc-Pro-Ala-Lys (Boc)-Lys (Boc-Pro-Ala-Lys)-Asp[OCH
2(CH
2)
10CH
3]-OCH
2(CH
2)
10CH
3Make 83mg (90%) target compound, be colourless powder.Mp?142℃;[α]
D 25=-55(c=0.72,H
2O);ESI-MS(m/e):1190[M+H]
+;IR(KBr):3225,3200,3186,3173,3048,3039,3015,2926,2855,1734,1653,1614,1549,1531,1456,1377,1362,1306,1287,1254,1206,1169.
1H?NMR(300MHz,DMSO-d
6):δ/ppm=8.82-8.80(m,2H),8.44-8.42(m,2H),8.18-8.16(m,2H),8.08-7.95(m,5H),4.63-4.61(m,1H),4.35(s,2H),4.19(s,5H),4.01(s,4H),3.20-3.17(m,5H),3.00(s,3H),2.74(s,6H),2.39-2.28(m,4H),2.14-1.99(m,1H),1.88-1.83(m,6H),1.62-1.52(m,13H),1.38-1.19(m,47H),0.86-0.83(m,6H).
Embodiment 38 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[OCH
2(CH
2)
12CH
3]-OCH
2-(CH
2)
12CH
3(6d)
According to the method for embodiment 7 by 150mg (0.09mmol) Boc-Pro-Ala-Lys (Boc)-Lys (Boc-Pro-Ala-Lys)-Asp[OCH
2(CH
2)
12CH
3]-OCH
2(CH
2)
12CH
3Make 119mg (94%) target compound, be colourless powder.Mp?144℃;[α]
D 25=-114(c=0.34,H
2O);ESI-MS(m/e):1246[M+H]
+;IR(KBr):3275,3202,3183,3169,3075,3053,2926,2855,1734,1651,1614,1545,1520,1504,1456,1290,1252,1238,1173.
1HNMR(500MHz,DMSO-d
6):δ/ppm=10.05-10.00(m,1H),8.85-8.84(d,J=7.5Hz,1H),8.57-8.39(m,1H),8.18-7.96(m,3H),4.64-4.62(m,1H),4.36-4.34(m,2H),4.23-4.17(m,5H),4.08-3.96(m,4H),3.62-3.44(m,1H),3.22-3.19(m,4H),3.06-2.94(m,2H),2.73-2.71(m,5H),2.31-2.29(m,2H),1.92-1.85(m,7H),1.79-1.64(m,4H),1.61-1.54(m,11H),1.36-1.24(m,60H),0.87-0.84(t,J=6.0Hz,J=7.0Hz,6H).
Embodiment 39 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[OCH
2(CH
2)
14CH
3]-OCH
2(CH
2)
14CH
3(6e)
According to the method for embodiment 7 by 185mg (0.11mmol) Boc-Pro-Ala-Lys (Boc)-Lys (Boc-Pro-Ala-Lys)-Asp[OCH
2(CH
2)
14CH
3]-OCH
2(CH
2)
14CH
3Make 150mg (95%) target compound, be colourless powder.Mp?145C;[α]
D 25=-97(c=0.37,H
2O);ESI-MS(m/e):1302[M+H]
+;IR(KBr):3302,3215,3208,3198,3051,2924,2853,2822,1736,1653,1545,1535,1456,1342,1308,1256,1242,1169.
1H?NMR(500MHz,DMSO-d
6):δ/ppm=10.03(m,1H),8.84(s,1H),8.57-8.50(m,1H),8.43-7.98(m,5H),4.64-4.63(d,J=5.0Hz,1H),4.36-4.32(m,2H),4.23-4.15(m,5H),4.04-3.98(m,4H),3.57-3.48(m,4H),3.23-3.19(m,5H),3.03-2.98(m,2H),2.73(s,6H),2.36-2.30(m,2H),1.91-1.86(m,7H),1.63-1.54(m,15H),1.38-1.24(m,67H),0.86-0.84(t,J=6.0Hz,J=7.0Hz,6H).
Embodiment 40 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[OCH
2(CH
2)
16CH
3]-OCH
2-(CH
2)
16CH
3(6f)
According to the method for embodiment 7 by 105mg (0.06mmol) Boc-Pro-Ala-Lys (Boc)-Lys (Boc-Pro-Ala-Lys)-Asp[OCH
2(CH
2)
16CH
3]-OCH
2(CH
2)
16CH
3Make 82mg (91%) target compound, be colourless powder.Mp?147℃;[α]
D 25=-181(c=0.12,H
2O);ESI-MS(m/e):1358[M+H]
+;IR(KBr):3427,3333,3208,2920,2851,2467,1929,1734,1622,1395,1336,1177,995,835,700,446.
1H?NMR(300MHz,DMSO-d
6):δ/ppm=8.80-8.75(m,1H),8.53-8.39(m,1H),8.06-7.94(m,2H),4.64-4.61(m,1H),4.36-4.34(m,2H),4.23-4.18(m,5H),4.03-3.97(m,3H),3.63(s,1H),3.54-3.49(m,2H),3.22-3.18(m,5H),3.01-2.97(m,2H),2.73(s,7H),2.31-2.29(m,3H),1.91-1.83(m,8H),1.74-1.53(m,28H),1.27-1.23(m,72H),0.87-0.83(t,J=5.1Hz,J=6.6Hz,6H).
Thrombus dissolving activity experiment in the body of experimental example 1 The compounds of this invention 6a-f
(6ml/kg i.p.) anaesthetizes with 20% urethane solution with the 200-220g male SD rat.The anesthetized rat dorsal position is fixed, and separates right common carotid artery, in proximal part folder bulldog clamp, proximal part and distal end penetrate surgical thread respectively, the surgical thread of distal end are clamped with mosquito forceps in fur, in the distal end intubate, unclamp bulldog clamp, emit about 1ml arterial blood and be contained in the EP pipe of 1ml.The Glass tubing of past vertical fixing (long 15mm, internal diameter 2.5mm, external diameter 5.0mm, the pipe end, seal with plug) and the middle 0.1ml of injection rat artery blood, the rapid thrombus standing bolt that inserts a stainless steel material in past the pipe.This thrombus fixedly spiral diameter is the Stainless Steel Wire coiled of 0.2mm, and the long 12mm of spiral part contains 15 bung flanges, and the diameter of bung flange is 1.0mm, and the holder handle links to each other with spiral, and long 7.0mm is the question mark type.Behind the blood coagulation 15min, open the plug of Glass tubing bottom, with the fixing fixing holder handle of spiral of thrombus of tweezers, the thrombus that taking-up is wrapped up by thrombus from Glass tubing is spiral fixedly, accurately weighs.
The bypass intubate constitutes by 3 sections, and the stage casing is polyethylene rubber tube, long 60mm, internal diameter 3.5mm, two ends are identical polyethylene tube, long 100mm, internal diameter 1mm, external diameter 2mm, one end of this pipe pulls into point pipe (being used for inserting rat carotid artery or vein), external diameter 1mm, the outer cover one segment length 7mm of the other end, the polyethylene tube of external diameter 3.5mm (overstriking is used for inserting in the polyethylene rubber tube in stage casing).The equal silanization of the inwall of 3 sections pipes.With the thrombus of thrombus parcel fixedly spiral put into the stage casing polyethylene rubber tube, the two ends of sebific duct are nested with two poly butt ends that add respectively.Standby with filling with heparin-saline solution (50IU/kg) in the pipe by sharp pipe end with syringe.
The left external jugular vein that separates rat, proximal part and distal end penetrate surgical thread respectively, on the left external jugular vein that exposes, cut an angle carefully, the sharp pipe of the bypass duct for preparing is above inserted the proximal part of left external jugular vein opening by angle, simultaneously away from the fixing holder handle of spiral of the interior thrombus in bypass tube stage casing (containing fixedly spiral of the thrombus of accurately weighing).Push the heparin-saline (50IU/kg) of accurate amount with syringe by the sharp pipe of the other end, this moment, syringe was not withdrawn polyethylene tube, clamped flexible pipe between syringe and the polyethylene tube with mosquito forceps.Proximal part in right common carotid artery stops blooding with bulldog clamp, right common carotid artery is being cut an angle carefully nearby from bulldog clamp.Extract syringe from the tip of polyethylene tube, the tip of polyethylene tube is inserted the proximal part of artery angle.The two ends of bypass duct all use 4 trumpeter's art sutures and arteriovenous to fix.
With scalp acupuncture with physiological saline (3ml/kg), the stage casing of the normal saline solution of the normal saline solution of urokinase (20000IU/kg) or 1nmol/kg 6a-f (embodiment 35-40 is prepared) by bypass tube (containing fixedly spiral of the thrombus of accurately weighing), thrust away from the fixing nearly vein place of spiral of thrombus, open bulldog clamp, make blood flow flow to vein by bypass duct from artery, this is rat arteriovenous shut Thrombolysis Model, slowly the liquid in the syringe is injected into (about 6min) in the blood, make physiological saline (blank), urokinase (positive control) or compound 6a-f of the present invention press the sequential action of vein-heart-artery to thrombus by blood circulation.Timing during from start injection, behind the 1h from bypass duct the fixing spiral of removal of thromboses, accurately weigh.Calculate fixedly of poor quality before and after the spiral administration of thrombus in every rat bypass duct, thrombolysis activity in the body of statistics and assessing compound.The result shows that 6a-f has thrombolysis activity (table 1) in the outstanding body.
The influence of the rat suppository loss of weight of table 1 1nmol/kg The compounds of this invention 6a-f
a
A) n=12, the thrombus loss of weight is represented with mean value SD mg; B) with physiological saline group ratio, p<0.01.
Experimental example 2 dosage are to the influence of thrombus dissolving activity in the The compounds of this invention 6d body
According to the experimental technique of experimental example 1, choose the thrombolysis activity under the best 6d of thrombolysis effect (embodiment 38 is prepared) investigation 1nmol/kg, 0.01nmol/kg and three dosage of 0.0001nmol/kg.The result shows, the thrombolytic effect show dose dependency (table 2) of The compounds of this invention 6d.
The dosage of table 26d is to the influence of rat suppository loss of weight
a
A) n=10, the thrombus loss of weight is represented with mean value SD mg; B) with physiological saline and 0.01 nmol/kg 6d group ratio, p<0.01; C) with physiological saline and 0.0001nmol/kg 6d group ratio, p<0.01; D) compare p>0.05 with physiological saline.
The external thrombus dissolving activity experiment of experimental example 3 The compounds of this invention 6a-f
1) making of thrombosis device
With internal diameter 4mm, external diameter 5.5mm, one section Glass tubing of length 18mm is placed on the quick detachable base of plastics, and the seam crossing of Glass tubing and plastic feet seals with one section emulsion tube.Place a Stainless Steel Wire spiral in the Glass tubing, screw diameter 1mm, length 20mm, the long hook of 2mm that comprises an end, blood namely be set in the stainless steel spiral around, thrombus can be hung up when weighing, when hatching, thrombus can be hung in the solution of reaction flask, do not run into wall, in order to avoid the damage thrombus.
2) making of reaction flask
10ml cillin bottle with the band rubber plug, wear a Stainless Steel Wire at rubber plug, end in bottle curves hook, thrombus hangs on the hook, be suspended in bottle interior testing compound solution, Stainless Steel Wire can move up and down at rubber plug, regulates the height of thrombus in solution, and it just is immersed in the solution to be measured.The simulation of internal milieu: estimate that according to the rat mean body weight every rat has 13ml blood, if rat thrombus in vivo model, blood that may about 8ml can touch thrombus, so add 8ml solution to be measured in the reaction flask, hatches at 37 ℃ of constant temperature shaking tables.
3) preparation of thrombus
With the 350-400g male SD rat with 20% urethane (6ml/kg, i.p.), anesthesia, it is fixing to lie on the back, and separates right common carotid artery, the bulldog clamp folder closes proximal part, the long polyethylene tube of 30mm is inserted in the bulldog clamp top, emits about 3-4ml blood at every turn, approximately can put 2-3 time, 5ml syringe with silanization injects the blood of emitting the Glass tubing that the preparation thrombus is used immediately one by one, the stainless steel spiral is put at once.Leave standstill 40min and make thrombosis, afterwards Glass tubing is carefully taken off from base, with fine needle with around the thrombus and the Glass tubing inwall separate, removal of thromboses hangs on the rubber plug of reaction flask, add 8ml distilled water in the reaction flask, thrombus is suspended on leaves standstill 1 hour in the water, remove the floating blood in thrombus surface.After 1 hour, inhale the moisture on the bolt surface of dehematizing with filter paper, accurately weigh one by one.
4) measure the external thrombolysis activity of 6a-f:
In each reaction flask, refill the normal saline solution of 6a-f (10nM), with physiological saline as blank, urokinase (100IU/ml) hangs on thrombus in the solution of The compounds of this invention 6a-f to be measured as positive control again, and 37 ℃ of constant temperature shaking table 70rpm were hatched 2 hours.After hatching end, draw surface water with filter paper and accurately weigh one by one again, calculate thrombus at the weight difference that adds solution to be measured front and back, the external thrombolysis activity of statistical appraisal compound.The result shows that The compounds of this invention 6a-f has outstanding external thrombolysis activity (table 3).
The external thrombolysis activity of table 310nM 6a-f
a
A) n=6, the thrombus loss of weight is represented with mean value SD mg; B) with physiological saline group ratio, p<0.01.
Experimental example 4 concentration are to the influence experiment of the external thrombus dissolving activity of The compounds of this invention 6b
According to the experimental technique of experimental example 3, choose the best 6b of thrombolysis effect (embodiment 36 is prepared) and investigate 10nM, the thrombolysis activity under three concentration of 1nM and 0.1nM.The result shows, the external thrombolytic effect display density dependency (table 4) of 6b.
The concentration of table 26b is to the influence of external thrombolysis activity
a
A) n=6, the thrombus loss of weight is represented with mean value SD mg; B) with physiological saline and 1nM 6b group ratio, p<0.01; C) with physiological saline and 0.1nM 4b group ratio, p<0.01; D) with physiological saline group ratio, p>0.05.
The nanometer ball experiment of experimental example 5 The compounds of this invention 6a-f
1) particle diameter of 6a-f nanometer ball in the aqueous solution
Recording the micelle-forming concentration of The compounds of this invention 6a-f in the aqueous solution earlier is 1 * 10
-12Mg/ml.Measure 6a-f 1 * 10 at Nano-ZS90 nano particle size determinator then
-12The particle diameter of mg/ml.The result shows that 6a-f can be assembled into nanometer ball in the aqueous solution, and particle diameter is 147 to 260nm (table 5).
The particle diameter of the nanometer ball that table 5 The compounds of this invention 6a-f assembles in the aqueous solution
2) form of the nanometer ball of 6a-f
It is 1 * 10 that The compounds of this invention 6a-f is made into concentration
-12The aqueous solution of mg/ml, then with this drips of solution on copper mesh, under the JEM-1230 transmission electron microscope, observe the form of nanometer ball behind the dried solvent that volatilizees.Mensuration shows, the nanometer ball of 6a-f formation rule.The transmission electron microscope photo of 6b is as representative (Fig. 3).
Claims (10)
2. the method for the described oligopeptides of synthetic claim 1 may further comprise the steps:
1) Boc-Pro and Ala-OBzl condensation are Boc-Pro-Ala-OBzl;
2) be Boc-Pro-Ala with the Boc-Pro-Ala-OBzl hydrogenolysis;
3) Boc-Pro-Ala and Lys (Boc)-OBzl condensation is Boc-Pro-Ala-Lys (Boc)-OBzl;
4) be Boc-Pro-Ala-Lys (Boc) with Boc-Pro-Ala-Lys (Boc)-OBzl hydrogenolysis;
5) Boc-Asp and saturated fatty alcohol condensation are Boc-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3, n=6,8,10,12,14 or 16 wherein;
6) Boc-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Slough Boc and generate Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3;
7) Boc-Lys (Boc) and Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Condensation is Boc-Lys (Boc)-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3;
8) Boc-Lys (Boc)-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Take off Boc and generate Lys-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3;
9) Boc-Pro-Ala-Lys (Boc) and Lys-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Condensation is Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3;
10) Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2(CH
2) nCH
3OCH
2(CH
2) nCH
3Remove Boc.
3. it is characterized in that in accordance with the method for claim 2: in the step 1) at dicyclohexyl carbonyl diimine and N-hydroxy benzo three
NitrogenIt is Boc-Pro-Ala-OBzl with the Ala-OBzl condensation in anhydrous THF that there is down Boc-Pro in azoles; Step 2) in methyl alcohol, be Boc-Pro-Ala with the Boc-Pro-Ala-OBzl hydrogenolysis in.
4. in accordance with the method for claim 2, it is characterized in that step 3) Boc-Pro-Ala in the presence of DCC and HOBt is Boc-Pro-Ala-Lys (Boc)-OBzl with Lys (Boc)-OBzl condensation in anhydrous THF.
5. in accordance with the method for claim 2, it is characterized in that step 4) is Boc-Pro-Ala-Lys (Boc) with Boc-Pro-Ala-Lys (Boc)-OBzl hydrogenolysis in methyl alcohol; In the step 5) in the presence of DCC Boc-Asp in anhydrous THF, be Boc-Asp[OCH with the saturated fatty alcohol condensation
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3
6. in accordance with the method for claim 2, it is characterized in that step 6) Boc-Asp[OCH in hydrogenchloride-ethyl acetate solution
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Take off Boc and generate Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3
7. in accordance with the method for claim 2, it is characterized in that step 7) in the presence of DCC and HOBt Boc-Lys (Boc) in anhydrous THF with Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Condensation is Boc-Lys (Boc)-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3
8. in accordance with the method for claim 2, it is characterized in that step 8) Boc-Lys (Boc)-Asp[OCH in hydrogenchloride-ethyl acetate solution
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Remove Boc and generate Lys-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3
9. in accordance with the method for claim 2, it is characterized in that step 9) in the presence of DCC and HOBt Boc-Pro-Ala-Lys (Boc) in anhydrous THF with Lys-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3Condensation is Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[OCH
2(CH
2) nCH
3]-OCH
2(CH
2) nCH
3, step 10) is Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc) in hydrogenchloride-ethyl acetate solution]-Asp[OCH
2(CH
2) nCH
3]-OCH
2-(CH
2) nCH
3Remove Boc.
10. the described oligopeptides of claim 1 is in the purposes of preparation in the thrombolytic agent.
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CN101190941A (en) * | 2006-11-30 | 2008-06-04 | 首都医科大学 | Polypeptide with thrombus dissolving activity and its preparation method and application |
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CN101190941A (en) * | 2006-11-30 | 2008-06-04 | 首都医科大学 | Polypeptide with thrombus dissolving activity and its preparation method and application |
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