CN102477074B - Oligopeptide compound and preparation method and application thereof - Google Patents
Oligopeptide compound and preparation method and application thereof Download PDFInfo
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- CN102477074B CN102477074B CN 201010573791 CN201010573791A CN102477074B CN 102477074 B CN102477074 B CN 102477074B CN 201010573791 CN201010573791 CN 201010573791 CN 201010573791 A CN201010573791 A CN 201010573791A CN 102477074 B CN102477074 B CN 102477074B
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Abstract
The invention relates to an oligopeptide with thrombus dissolving activities and a preparation method and application thereof. The oligopeptide is shown in the general formula 6a-f in the specification, wherein in the formula, n is 6, 8, 10, 12, 14 or 16. Through animal experiments, the in vitro and in vivo thrombus dissolving activities of the oligopeptide are evaluated, and the oligopeptide is proved to have excellent thrombus dissolving activities and be used for preparing thrombus dissolving drugs. The general formula 6a-f is shown in the specification.
Description
Technical field
The present invention relates to have the oligopeptide compounds of thrombus dissolving activity, belonged to biomedicine 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 amide 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
The object of the present invention is to provide the oligopeptide compounds with thrombus dissolving activity, and estimated the thrombus dissolving activity of these compounds by experimentation on animals, proved that oligopeptide compounds of the present invention has outstanding thrombus dissolving activity.
The present invention is achieved through the following technical solutions:
One of purpose of the present invention provides the compound of following structure,
Wherein, n is 6,8,10,12,14 or 16.
Two of the object of the invention provides the method for the above compound of preparation, specifically comprises the steps:
1) in the presence of dicyclohexylcarbodiimide (DCC) and N-hydroxy-succinamide (HOSu), Boc-Pro is Boc-Pro-Ala with the Ala condensation in anhydrous THF;
2) in anhydrous THF, be Boc-Pro-Ala-Lys (Z)-OBzl or Boc-Pro-Ala-Lys-(Boc)-OBzl with Lys (Z)-OBzl or Lys-(Boc)-OBzl condensation at Boc-Pro-Ala in the presence of DCC and the HOBt;
3) in methyl alcohol, with step 2) the product saponification that obtains is Boc-Pro-Ala-Lys (Z) or Boc-Pro-Ala-Lys-(Boc);
4) Boc-Asp is Boc-Asp[NHCH with the condensation of saturated fatty amine in anhydrous THF in the presence of DCC
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3, wherein, n is 6,8,10,12,14 or 16;
5) at hydrogenchloride-ethyl acetate solution Boc-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3Slough Boc and generate Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3
6) Boc-Lys (Boc) in the presence of DCC and the HOBt in anhydrous THF with
Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3Condensation is Boc-Lys (Boc)-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3
7) Boc-Lys (Boc)-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3In hydrogenchloride-ethyl acetate solution, remove Boc and generate Lys-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3
8) in the presence of DCC and HOBt, the step 3) products therefrom in anhydrous THF with Lys-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3Condensation is
Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3Or
Boc-Pro-Ala-Lys(Boc)-Lys[Boc-Pro-Ala-Lys(Boc)]-Asp[NHCH
2(CH
2)nCH
3]-NHCH
2(CH
2)nCH
3；
9) condensation product when step 8) is Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3The time, earlier with the product of step 8) at methyl alcohol, Pd/C and H
2Following hydrogenolysis is
Boc-Pro-Ala-Lys-Lys[Boc-Pro-Ala-Lys]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3, again will
Boc-Pro-Ala-Lys-Lys[Boc-Pro-Ala-Lys]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3In hydrogenchloride-ethyl acetate solution, remove Boc and generate the described compound of claim 1;
When the condensation product of step 8) is
Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3The time, will
Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3In hydrogenchloride-ethyl acetate solution, remove Boc and generate The compounds of this invention.
Three of purpose of the present invention provides a kind of pharmaceutical composition, and this pharmaceutical composition contains the The compounds of this invention that effective dose is gone up in treatment, and contains one or more pharmaceutically acceptable excipient or auxilliaryly add agent.
Four of purpose of the present invention provides a kind of pharmaceutical preparation, and this pharmaceutical preparation is with The compounds of this invention and pharmaceutically acceptable excipient or assists the mixture that adds agent and make tablet, capsule, pulvis, granule, lozenge or oral liquid.
Compound of the present invention and pharmaceutical composition of the present invention all can be used for preparing thrombolytic agent.
The present invention has estimated thrombus dissolving activity in the external and body of The compounds of this invention by experimentation on animals, experimental result proves that compound of the present invention has outstanding thrombus dissolving activity, can be used for preparing thrombolytic agent.
Description of drawings
Fig. 1 is the structural formula of The compounds of this invention;
Fig. 2 is the synthetic route chart of The compounds of this invention;
Fig. 3 is the transmission electron microscope photo of The compounds of this invention 6c.
In Fig. 2, i) DCC, HOBt and NMM; Ii) NaOH; Iii) HCl/EA; Iv) Pd/C; H
2
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.
In an embodiment of the present invention, following implication represented in used breviary term:
The THF tetrahydrofuran (THF)
HOBt N-hydroxy benzo triazole
The DCC dicyclohexylcarbodiimide
The DCU dicyclohexylurea (DCU)
The TLC thin-layer chromatography
The Boc tertbutyloxycarbonyl
Embodiment 1 preparation Boc-Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3(1a)
1.17g (5mmol) Boc-Asp is dissolved with the anhydrous THF of 20ml.In the solution that obtains, add 0.68g (5.0mmol) N-hydroxy benzo triazole (HOBt).After 10 minutes, under ice bath, add the solution of 2.06g (10mmol) dicyclohexylcarbodiimide (DCC) and the anhydrous THF of 25ml, obtain reaction solution (I).1.42g (11mmol) NH
2CH
2(CH
2)
6CH
3Be 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, TLC (methylene chloride, 20: 1) shows that Boc-Asp disappears.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.The ethyl acetate solution anhydrous Na
2SO
4Drying, filtration, filtrate decompression are concentrated into dried, and residue obtains 1.640g (72%) target compound through column chromatography purification, is colourless powder.ESI-MS(m/e)：456[M+H]
+.
Embodiment 2 preparation Boc-Asp[NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3(1b)
According to the method for embodiment 1 by 2.8g (12mmol) Boc-Asp and 4.15g (26.4mmol) CH
3(CH
2)
8CH
2NH
2Make 4.353g (71%) target compound, be colourless powder.ESI-MS(m/e)：512[M+H]
+.
Embodiment 3 preparation Boc-Asp[NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3(1c)
According to the method for embodiment 1 by 5.0g (21.5mmol) Boc-Asp and 9.58g (51.50mmol) CH
3(CH
2)
10CH
2NH
2Make 7.94g (65%) target compound, be colourless powder.ESI-MS(m/e)：568[M+H]
+.
Embodiment 4 preparation Boc-Asp[NHCH
2(CH
2)
12CH
3]-NHCH
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
2NH
2Make 8.32g (62%) target compound, be colourless powder.ESI-MS(m/e)：624[M+H]
+.
Embodiment 5 preparation Boc-Asp[NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3(1e)
According to the method for embodiment 1 by 2.78g (12mmol) Boc-Asp and 6.36g (26.4mmol) CH
3(CH
2)
14CH
2NH
2Make 3.74g (46%) target compound, be colourless powder.ESI-MS(m/e)：680[M+H]
+.
Embodiment 6 preparation Boc-Asp[NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3(1f)
According to the method for embodiment 1 by 2.33g (10mmol) Boc-Asp and 6.73g (25mmol) CH
3(CH
2)
16CH
2NH
2Make 3.61g (49%) target compound, be colourless powder.ESI-MS(m/e)：736[M+H]
+.
Embodiment 7 preparation Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3(2a)
With 2.28g (5.0mmol) Boc-Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3Be dissolved in 20ml 4N hydrogenchloride-ethyl acetate solution, stirring at room 1 hour, TLC (methylene chloride, 20: 1) shows Boc-AspAsp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3Disappear, 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 1.62g (93%) target compound, be colourless powder.ESI-MS(m/e)：356[M+H]
+.
Embodiment 8 preparation Asp[NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3(2b)
Method 2.044g (4.00mmol) Boc-Asp[NHCH according to embodiment 7
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3Make 1.72g (84%) target compound, be colourless powder.ESI-MS(m/e)：412[M+H]
+.
Embodiment 9 preparation Asp[NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3(2c)
Method 6.28g (11.0mmol) Boc-Asp[NHCH according to embodiment 7
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3Make 5.24g (94%) target compound, be colourless powder.ESI-MS(m/e)：468[M+H]
+.
Embodiment 10 preparation Asp[NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3(2d)
Method 6.32g (10.1mmol) Boc-Asp[NHCH according to embodiment 7
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3Make 5.39g (95%) target compound, be colourless powder.ESI-MS(m/e)：524[M+H]
+.
Embodiment 11 preparation Asp[NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3(2e)
Method 2.72g (4.0mmol) Boc-Asp[NHCH according to embodiment 7
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3Make 2.24g (82%) target compound, be colourless powder.ESI-MS(m/e)：580[M+H]
+.
Embodiment 12 preparation Asp[NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3(2f)
Method 3.68g (5.0mmol) Boc-Asp[NHCH according to embodiment 7
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3Make 2.86g (90%) target compound, be colourless powder.ESI-MS(m/e)：636[M+H]
+.
Embodiment 13 preparation Boc-Lys (Boc)-Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3(3a)
According to the method for embodiment 1 by 1.26g (3.6mmol) Boc-Lys (Boc) and 1.18g (3.0mmol) Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3Make 1.63g (80%) target compound, be colourless powder.ESI-MS(m/e)：685[M+H]
+.
Embodiment 14 preparation Boc-Lys (Boc)-Asp[NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3(3b)
According to the method for embodiment 1 by 1.246g (3.60mmol) Boc-Lys (Boc) and 1.34g (3.0mmol) Asp[NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3Make 1.65g (74%) target compound, be colourless powder.ESI-MS(m/e)：741[M+H]
+.
Embodiment 15 preparation Boc-Lys (Boc)-Asp[NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3(3c)
According to the method for embodiment 1 by 1.25g (3.6mmol) Boc-Lys (Boc) and 1.51g (3.0mmol) Asp[NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3Make 1.72g (72%) target compound, be colourless powder.ESI-MS(m/e)：797[M+H]
+.
Embodiment 16 preparation Boc-Lys (Boc)-Asp[NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3(3d)
According to the method for embodiment 1 by 1.25g (3.6mmol) Boc-Lys (Boc) and 1.68g (3.0mmol) Asp[NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3Make 1.72g (67%) target compound, be colourless powder.ESI-MS(m/e)：853[M+H]
+.
Embodiment 17 preparation Boc-Lys (Boc)-Asp[NHCH
2(CH
2)
14CH
3]-NHCH
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[NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3Make 1.85g (68%) target compound, be colourless powder.ESI-MS(m/e)：909[M+H]
+.
Embodiment 18 preparation Boc-Lys (Boc)-Asp[NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3(3f)
According to the method for embodiment 1 by 0.42g (1.2mmol) Boc-Lys (Boc) and 0.67g (1.0mmol) Asp[NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3Make 0.81g (85%) target compound, be colourless powder.ESI-MS(m/e)：965[M+H]
+.
Embodiment 19 preparation Lys-Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3(4a)
Method 1.37g (2.0mmol) Boc-Lys (Boc)-Asp[NHCH according to embodiment 7
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3Make 0.87g (90%) target compound, be colourless powder.ESI-MS(m/e)：485[M+H]
+.
Embodiment 20 preparation Lys-Asp[NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3(4b)
Method 1.48g (2.0mmol) Boc-Lys (Boc)-Asp[NHCH according to embodiment 7
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3Make 0.99g (99%) target compound, be colourless powder.ESI-MS(m/e)：541[M+H]
+.
Embodiment 21 preparation Lys-Asp[NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3(4c)
Method 1.72g (2.2mmol) Boc-Lys ((Boc)-Asp[NHCH according to embodiment 7
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3Make 1.34g (93%) target compound, be colourless powder.ESI-MS(m/e)：597[M+H]
+.
Embodiment 22 preparation Lys-Asp[NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3(4d)
Method 1.72g (2.0mmol) Boc-Lys (Boc)-Asp[NHCH according to embodiment 7
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3Make 1.30g (89%) target compound, be colourless powder.ESI-MS(m/e)：653[M+H]
+.
Embodiment 23 preparation Lys-Asp[NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3(4e)
Method 1.83g (2.0mmol) Boc-Lys (Boc)-Asp[NHCH according to embodiment 7
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3Make 1.29g (90%) target compound, be colourless powder.ESI-MS(m/e)：709[M+H]
+.
Embodiment 24 preparation Lys-Asp[NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3(4f)
Method 1.93g (2.0mmol) Boc-Lys (Boc)-Asp[NHCH according to embodiment 7
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3Make 1.39g (91%) target compound, be colourless powder.ESI-MS(m/e)：765[M+H]
+.
Embodiment 25 preparation Boc-Pro-Ala
2.7g (12.6mmol) Boc-Pro with the anhydrous THF dissolving of 30ml, is added 1.59g (13.86mmol) N-hydroxy-succinamide (HOSu) in the solution that obtains.After 10 minutes, under ice bath, add 2.86g (13.9mmol) DCC and the anhydrous THF of 20ml.At room temperature stir 10h, TLC (methylene chloride, 30: 1) shows that Boc-Pro disappears, the reaction solution filtering DCU that obtains, and THF is removed in decompression.Residue is with using 5%NaHCO after the 250ml acetic acid ethyl dissolution
3It is inferior to give a baby a bath on the third day after its birth, and collects organic phase, and ethyl acetate is removed in decompression, and the colorless solid that obtains dissolves with THF, gets reaction solution (I).1.23g (13.9mmol) Ala is dissolved in 30ml water obtains reaction solution (II).Saturated NaHCO will also be used under the ice bath in reaction solution (II) the adding reaction solution (I)
3Transfer pH to 8, stirring at room 16h then, TLC (methylene chloride, 30: 1) shows that the Boc-Pro-OSu in the reaction solution (I) disappears.The saturated KHSO of reaction solution
4Transfer pH to 7, decompression is except THF, the saturated KHSO of residue
4Transfer pH to 2.The solution that obtains is with ethyl acetate extraction 5 times, and the organic phase of collection is given a baby a bath on the third day after its birth with the saturated NaCl aqueous solution and used anhydrous Na after inferior
2SO
4Drying, filtration, filtrate decompression are concentrated into the dried 3.44g of obtaining (95%) target compound, are colourless powder.ESI-MS(m/e)：287[M+H]
+.
Embodiment 26 preparation Boc-Pro-Ala-Lys (Z)-OBzl
Method according to embodiment 1 makes 1.56g (61%) target compound by 1.37g (4.8mmol) Boc-Pro-Ala and 2.17g (4.0mmol) Lys (Z)-OBzl, is colourless powder.ESI-MS(m/e)：639[M+H]
+.
Embodiment 27 preparation Boc-Pro-Ala-Lys (Boc)-OBzl
Method according to embodiment 1 makes 4.47g (85%) target compound by 3.15g (11.0mmol) Boc-Pro-Ala and 2.97g (10.0mmol) Lys (Boc)-OBzl, is colourless powder.ESI-MS(m/e)：529[M+H]
+.
Embodiment 28 preparation Boc-Pro-Ala-Lys (Z)
1.92g (3.0mmol) Boc-Pro-Ala-Lys (Z)-OBzl is dissolved in the methyl alcohol, and ice bath stirs down and transfers pH to 12 and stir 4h with 2N NaOH, TLC (methylene dichloride: methyl alcohol=30: 1) show that Boc-Pro-Ala-Lys (Z)-OBzl disappears.The saturated KHSO of reaction mixture
4Transfer pH to 6, decompression is except using saturated KHSO after the methyl alcohol again
4Transfer pH to 2, the mixture that obtains ethyl acetate extraction 5 times, the organic phase of collection is used anhydrous Na after washing 3 times with the saturated NaCl aqueous solution
2SO
4Drying, filtration, filtrate decompression are concentrated into dried 1.48g (90%) target compound, are colourless powder.ESI-MS(m/e)：547[M-H]
-.
Embodiment 29 preparation Boc-Pro-Ala-Lys (Boc)
Method according to embodiment 28 makes 2.34g (93%) target compound by 2.64g (5.0mmol) Boc-Pro-Ala-Lys (Boc)-OBzl, is colourless powder.ESI-MS(m/e)：515[M-H]
-.
Embodiment 30 preparation Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3(5a)
According to the method for embodiment 1 by 1.32g (2.6mmol) Boc-Pro-Ala-Lys (Z) and 650mg (1.16mmol) Lys-Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3Make 180mg (11%) target compound, be colourless powder.116 ℃ of Mp; [α]
D 25=-48.8 (c=0.65, methyl alcohol); ESI-MS (m/e): 1545[M+H]
+IR (KBr): 3296.72,3080.40,2930.87,2863.14,1694.10,1642.96,1543.49,1452.23,1401.17,1254.75,1164.15,1119.18,1029.28,976.16,698.29.
1H NMR (300MHz, DMSO-d
6): 8.19-7.96 (m, 3H), 7.87-7.70 (m, 2H), 7.43-7.28 (m, 9H), 7.22-7.12 (m, 1H), 5.0 (s, 4H), 4.13-4.02 (m, 3H), 3.41-3.38 (m, 2H), 3.16 (s, 1H), and 3.06-2.87 (m, 9H), 2.14-2.02 (m, 2H), 1.87-1.68 (m, 6H), 1.45-1.28 (m, 26H), 1.28-1.12 (m, 29H), 0.85 (d, J=6.3Hz, 6H).
Embodiment 29 preparation Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3(5b)
According to the method for embodiment 1 by 1.138g (1.0mmol) Boc-Pro-Ala-Lys (Boc) and 0.494g (1mmol) Lys-Asp[NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3Make 0.139g (9%) target compound, be colourless powder.103 ℃ of Mp; [α]
D 25=-38.9 (c=0.55, methyl alcohol); ESI-MS (m/e): 1533[M+H]
+IR (KBr): 3417,3357,3067,2926,2857,2741,1650,1549,1458,1380,1297,1237,1107,1056,989,937,897,849,735,550.
1H NMR (300MHz, DMSO-d
6): 8.19-7.94 (m, 4H), 7.94-7.59 (m, 4H), 7.57-7.36 (m, 2H), 6.70 (s, 2H), 4.51-4.02 (m, 8H), 3.71-3.58 (m, 2H), 3.12-2.92 (m, 7H), 2.92-2.78 (m, 5H), 2.35 (s, 1H), 2.16-1.97 (m, 2H), 1.94-1.55 (m, 10H), 1.55-1.28 (m, 49H), 1.28-1.09 (m, 39H), 0.85 (t, J=5.4Hz, J=6.6Hz, 6H).
Embodiment 30 preparation Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3(5c)
According to the method for embodiment 1 by 1.182g (2.3mmol) Boc-Pro-Ala-Lys (Z) and 0.70g (1.04mmol) Lys-Asp[NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3Make 0.21g (13%) target compound, be colourless powder.Mp?197℃；ESI-MS(m/e)：1658[M+H]
+.
1H?NMR(300MHz，DMSO-d
6)：δ/ppm＝8.02-7.83(m，5H)，7.33(s，9H)，7.19(s，2H)，4.99(s，4H)，4.41-4.30(m，4H)，4.19-4.01(m，4H)，2.97(s，10H)，2.08(s，2H)，1.98(s，3H)，1.78(s，8H)，1.51-1.49(m，3H)，1.38(s，11H)，1.31(s，16H)，1.225(s，48H)，0.85-0.83(t，J＝6.0Hz?J＝6.6Hz?6H).
Embodiment 31 preparation Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3(5d)
According to the method for embodiment 1 by 1.167g (2.27mmol) Boc-Pro-Ala-Lys (Z) and 750mg (1.03mmol) Lys-Asp[NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3Make 0.313g (18%) target compound, be colourless powder.Mp?106℃；IR(KBr)：3323.64，2927.85，2853.85，2687.75，2614.76，2475.82，1691.03，1635.55，1538.16，1454.79，1398.84，1311.14，1245.16，119.11，1113.40，1045.63，989.39，890.35，767.76，648.93，538.93，466.01，415.94.ESI-MS(m/e)：1713[M+H]
+.
1H?NMR(300MHz，DMSO-d
6)：δ/ppm＝8.1-8.0(m，2H)，7,88-7.62(m，7H)，7.34(s，13H)，7.19(s，2H)，4.99(s，4H)，4.11-4.04(m，8H)，2.95-2.94(m，14H)，2.07-1.99(m，4H)，1.78-1.76(m，10H)，1.61(s，4H)，1.39(s，17H)，1.35(s，3H)，1.31(s，19H)，1.23(s，56H)，0.87-0.83(t，J＝4.8Hz?J＝6.9Hz?6H).
Embodiment 32 preparation Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3(5e)
According to the method for embodiment 1 by 1.138g (1.0mmol) Boc-Pro-Ala-Lys (Boc) and 0.678g (1.1mmol) Lys-Asp[NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3Make 0.31g (18%) target compound, be colourless powder.108 ℃ of Mp; [α]
D 25=-25.3 (c=0.75, methyl alcohol); ESI-MS (m/e): 1701[M+H]
+IR (KBr): 3292,3087,2927,2857,1691,1641,1452,1390,1249,1169,1119,992,857,745,702.
1H NMR (300MHz, DMSO-d
6): δ/ppm=8.22-7.60 (m, 8H), 7.60-7.33 (m, 2H), 6.78-6.62 (s, 2H), 4.52-4.05 (m, 7H), 3.15-2.80 (m, 10H), 2.21-1.94 (m, 3H), and 1.94-1.62 (m, 8H), 1.62-1.05 (m, 92H), 0.92-0.78 (t, J=5.4Hz, J=6.6Hz, 6H).
Embodiment 33 preparation Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3(5f)
According to the method for embodiment 1 by 0.603g (1.1mmol) Boc-Pro-Ala-Lys (Z) and 0.418g (0.5mmol) Lys-Asp[NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3Make 0.23g (25%) target compound, be colourless powder.107 ℃ of Mp; [α]
D 25=-3.5 (c=0.6, methyl alcohol); ESI-MS (m/e): 1825[M+H]
+IR (KBr): 3297.55,3080.33,2922.96,2853.41,1695.84,1641.14,1545.33,1456.85,1400.91,1256.66,1164.52,705.96.
1H NMR (300MHz, DMSO-d
6): δ/ppm=8.17-7.92 (m, 5H), 7.90-7.63 (m, 5H), 7.40-7.38 (m, 14H), and 7.23-7.12 (m, 3H), 5.0 (s, 4H), and 4.52-4.05 (m, 11H), 3.09-2.82 (m, 13H), and 2.19-1.96 (m, 4H), 1.96-1.71 (m, 10H), and 1.71-1.28 (m, 51H), 1.28-1.0 (m, 71H), 0.94-0.78 (d, J=6.6Hz, 6H).
Embodiment 34 preparation Boc-Pro-Ala-Lys-Lys (Boc-Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3
With 0.771g (0.5mmol) Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3Use the 25ml dissolve with methanol, add 0.15g Pd/C, logical H
2(0.02Mba) and stirring at room to Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3Disappear.Filtering Pd/C, filtrate decompression are concentrated into dried 0.60g (94%) target compound, are colourless powder.ESI-MS(m/e)：1277[M+H]
+.
Embodiment 35 preparation Boc-Pro-Ala-Lys-Lys (Boc-Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3
According to the method for embodiment 34 by 0.828g (0.50mmol) Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3Make 0.638g (92%) target compound, be colourless powder.ESI-MS(m/e)：1389[M+H]
+.
Embodiment 36 preparation Boc-Pro-Ala-Lys-Lys (Boc-Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3
According to the method for embodiment 34 by 0.856g (0.50mmol) Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3Make 0.665g (92%) target compound, be colourless powder.ESI-MS(m/e)：1445[M+H]
+.
Embodiment 37 preparation Boc-Pro-Ala-Lys-Lys (Boc-Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3
According to the method for embodiment 34 by 0.912g (0.50mmol) Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)] Asp[NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3Make 0.725g (92%) target compound, be colourless powder.ESI-MS(m/e)：1577[M+H]
+.
Embodiment 38 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys) Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3(6a)
According to the method for embodiment 7 by 0.789g (0.5mmol) Boc-Pro-Ala-Lys-Lys (Boc-Pro-Ala-Lys) Asp[NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3Make 0.516g (96%) target compound, be colourless powder.103 ℃ of Mp; [α]
D 25=-38.9 (c=0.55, methyl alcohol); ESI-MS (m/e): 1077[M+H]
+IR (KBr): 3412,3278,3063,2931,2856,1655,1546,1546,1456,1381,1247,1165,1041,655.
1H NMR (300MHz, DMSO-d
6): 8.41-7.98 (m, 2H), 4.49-4.22 (m, 8H), 3.70-3.41 (m, 5H), and 3.26-3.04 (m, 7H), 3.04-2.91 (m, 5H), 2.78-2.67 (m, 2H), 2.47 (s, 2H), 2.18-2.00 (m, 7H), and 1.79-1.64 (m, 9H), 1.60-1.37 (m, 22H), 1.32 (s, 20H), 1.23-1.15 (m, 3H), 0.85 (t, J=5.1Hz, J=6.6Hz, 6H).
Embodiment 39 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3(6b)
According to the method for embodiment 7 by 0.120g (0.07mmol) Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3Make 0.083g (93%) target compound, be colourless powder.91 ℃ of Mp; [α]
D 25=-26.5 (c=0.55, methyl alcohol); ESI-MS (m/e): 1133[M+H]
+.IR (KBr): 3417,3392,3067,3377,2926,2857,2741,1650,1549,1459,1380,1335,1238,1108,1075,987,943,881,735,551.
1H NMR (300MHz, DMSO-d
6): δ/ppm=10.05 (s, 2H), 8.92-8.78 (d, J=6.6Hz, 2H), 8.58-8.42 (s, 2H), 8.37-8.13 (m, 2H), 8.13-7.88 (m, 10H), and 7.75-7.68 (d, J=8.4Hz, 2H), 7.50-7.58 (t, J=7.2Hz, J=7.5Hz, 2H), 7.47-7.38 (t, J=7.8Hz, J=7.5Hz, 2H), 4.53-4.32 (m, 3H), 4.29-4.08 (m, 5H), 3.98-3.89 (m, 2H), 3.83-2.68 (m, 5H), 3.13-2.88 (m, 9H), 2.82-2.67 (m, 7H), 2.38-2.22 (m, 2H), 1.95-1.82 (m, 6H), 1.44-1.15 (m, 43H), 0.85 (t, J=5.4Hz, J=6.9Hz, 6H).
Embodiment 40 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3(6c)
According to the method for embodiment 7 by 0.694g (0.50mmol) Boc-Pro-Ala-Lys-Lys (Boc-Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3Make 0.535g (90%) target compound, be colourless powder.109 ℃ of Mp; [α]
D 25=-41.56 (c=0.60, methyl alcohol); ESI-MS (m/e): 1189[M+H]
+.IR (KBr): 3287.90,3070.41,2924.07,2854.39,1644.63,1548.28,1460.31,1378.54,1294.89,1249.94,1164.13,1041.54,714.64.
1HNMR (300MHz, DMSO-d
6): δ/ppm=9.98 (s, 1H), 8.83 (s, 1H), 8.68 (s, 1H), 8.35-7.90 (m, 5H), 7.69 (s, 1H), 7.38 (s, 1H), 4.48-4.34 (m, 2H), 4.32-4.05 (m, 4H), 3.20 (s, 6H), 2.97 (s, 8H), 2.73 (s, 4H), 2.46-2.30 (m, 5H), 1.89 (s, 6H), 1.78 (s, 1H), 1.75-1.55 (m, 9H), 1.34 (s, 9H), 1.23 (s, 42H), 0.86-0.82 (t J=6.0Hz J=6.9Hz 6H).
Embodiment 41 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3(6d)
According to the method for embodiment 7 by 0.723g (0.50mmol) Boc-Pro-Ala-Lys-Lys (Boc-Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3Make 0.566g (91%) target compound, be colourless powder.108 ℃ of Mp; [α]
D 25=-43.69 (c=0.50, methyl alcohol); IR (KBr): 3326.16,3039.73,2925.81,2850.71,2360.91,1626.48,1574.44,1438.57,1311.61,1242.74,1087.61,1045.63,894.44,649.16,452.09.ESI-MS (m/e): 1245[M+H]
+.
1H NMR (300MHz, DMSO-d
6): δ/ppm=9.98 (s, 1H), 8.85-8.83 (d, J=6.0Hz 1H), 8,58 (s, 1H), 8.05 (s, 5H), 7.58-7.33 (m, 2H), 4.81-4.36 (m, 2H), 4.21-4.11 (m, 4H), 3.20 (s, 6H), 2.97 (s, 8H), 2.73 (s, 4H), 2.45-2.41 (m, 2H), 2.31-2.29 (m, 3H), 1.98-1.86 (m, 6H), 1.78 (s, 1H), 1.57-1.55 (m, 9H), 1.23 (s, 56H), 0.86-0.82 (t, J=6.0Hz, J=6.9Hz, 6H).
Embodiment 42 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3(6e)
According to the method for embodiment 7 by 0.120g (0.07mmol) Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3Make 0.089g (97%) target compound, be colourless powder.96 ℃ of Mp; [α]
D 25=-23.5 (c=0.60, water); ESI-MS (m/e): 1301[M+H]
+IR (KBr): 3411,3272,3068,2923,2854,1650,1549,1462,1736,1376,1267,1249,1149,1097,1062,549.
1H NMR (300MHz, DMSO-d
6): δ/ppm=8.92-8.80 (m, 2H), 8.55-7.83 (m, 10H), 7.67-7.58 (m, 2H), and 4.53-4.06 (m, 6H), 3.28-3.12 (m, 5H), 3.12-2.86 (m, 6H), 2.86-2.67 (m, 4H), 2.40-2.21 (m, 2H), 1.99-1.76 (m, 6H), 1.76-1.49 (m, 9H), 1.49-1.05 (m, 64H), 0.86-0.84 (t, J=3.9Hz, J=6.0Hz, 6H).
Embodiment 43 preparation Pro-Ala-Lys-Lys (Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3(6f)
According to the method for embodiment 7 by 0.639g (0.5mmol) Boc-Pro-Ala-Lys-Lys (Boc-Pro-Ala-Lys)-Asp[NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3Make 0.603g (89%) target compound, be colourless powder.105 ℃ of Mp; [α]
D 25=-18.5 (c=0.60, methyl alcohol); ESI-MS (m/e): 1357[M+H]
+IR (KBr): 3286.91,3064.09,2921.86,2852.60,1648.64,1546.83,1461.29,1376.50,1249.16,1162.49,1045.63,570.15.
1H NMR (300MHz, DMSO-d
6): δ/ppm=8.37-7.78 (m, 7H), 4.45-4.29 (m, 3H), 4.29-4.09 (m, 6H) 3.30-3.13 (d, J=2.5Hz, 7H), 3.02 (s, 7H), 2.81-2.67 (m, 5H), 2.39-2.18 (m, 3H), 1.99-1.78 (m, 8H), 1.72-1.48 (m, 13H), 1.48-1.12 (m, 74H), 0.95-0.79 (d, J=6.3Hz, 6H).
The thrombus dissolving activity of experimental example 16a-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 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 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 results are shown in Table 1.
The influence of the rat suppository loss of weight of table 11nmol/kg 6a-f
a
A) n=10, wet weight of thrombus is represented with mean value SD mg; B) compare p＜0.01 with the physiological saline group.
Experimental example 2 dosage are to the influence of thrombus dissolving activity in the 6c body
According to the experimental technique of experimental example 1, choose best 6c investigation 1nmol/kg, the 0.01nmol/kg of thrombolysis effect and the thrombolysis activity under three dosage of 0.001nmol/kg.The result is as shown in table 2.The result shows, the thrombolytic effect show dose dependency of 6c.
Table 2
Table 2 is that the dosage of 6c is to the influence of rat suppository loss of weight
a, wherein a represents sample number, i.e. the number of used rat, a) n=10; The thrombus loss of weight is represented with mean value SD mg; B) with physiological saline and 0.01nmol/kg 6c group ratio, p＜0.01; C) compare p＜0.01 with physiological saline and 0.001nmol/kg 6c; D) compare p＜0.05 with physiological saline.
The particle diameter of the nanometer ball of 6a-f in experimental example 2 aqueous solution
1) particle diameter of 6a-f nanometer ball in the aqueous solution
6a-f is 10 when the laser light scattering particle size analyzer has been observed 25 ℃ and 37 ℃
-4M, 10
-5M and 10
-6The particle diameter of the nanometer ball that forms in the M aqueous solution.The result is as shown in table 3, and the result shows that 6a-f can be assembled into nanometer ball in the aqueous solution, and particle diameter is 120 to 416nm in the time of 25 ℃, and particle diameter is 85 to 306nm in the time of 37 ℃.
Table 3
2) form of the nanometer ball of 6a-f
It is 1 * 10 that 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.Be example with 6c, the transmission electron microscope photo of 6c as shown in Figure 3.As can be seen from Figure 3, the nanometer ball of 6a-f formation rule.
Claims (6)
1. the compound of following structure,
Wherein, n is 6,8,10,12,14 or 16.
2. a method for preparing the described compound of claim 1 is characterized in that, comprises the steps:
1) in the presence of dicyclohexylcarbodiimide (DCC) and N-hydroxy-succinamide (HOSu), Boc-Pro is Boc-Pro-Ala with the Ala condensation in anhydrous THF;
2) in anhydrous THF, be Boc-Pro-Ala-Lys (Z)-OBzl or Boc-Pro-Ala-Lys-(Boc)-OBzl with Lys (Z)-OBzl or Lys-(Boc)-OBzl condensation at Boc-Pro-Ala in the presence of DCC and the HOBt;
3) in methyl alcohol, with step 2) the product saponification that obtains is Boc-Pro-Ala-Lys (Z) or Boc-Pro-Ala-Lys-(Boc);
4) Boc-Asp is Boc-Asp[NHCH with the condensation of saturated fatty amine in anhydrous THF in the presence of DCC
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3, wherein, n is 6,8,10,12,14 or 16;
5) Boc-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3In hydrogenchloride-ethyl acetate solution, slough Boc and generate Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3;
6) in the presence of DCC and HOBt, Boc-Lys (Boc) in anhydrous THF with Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3Condensation is Boc-Lys (Boc)-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3;
7) in hydrogenchloride-ethyl acetate solution, Boc-Lys (Boc)-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3Remove Boc and generate Lys-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3
8) in the presence of DCC and HOBt, the step 3) products therefrom in anhydrous THF with Lys-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3Condensation is
Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3Or
Boc-Pro-Ala-Lys(Boc)-Lys[Boc-Pro-Ala-Lys(Boc)]-Asp[NHCH
2(CH
2)nCH
3]-NHCH
2(CH
2)nCH
3；
9) condensation product when step 8) is
Boc-Pro-Ala-Lys (Z)-Lys[Boc-Pro-Ala-Lys (Z)]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3The time, earlier with the product of step 8) at methyl alcohol, Pd/C and H
2Following hydrogenolysis is
Boc-Pro-Ala-Lys-Lys[Boc-Pro-Ala-Lys]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3, again will
Boc-Pro-Ala-Lys-Lys[Boc-Pro-Ala-Lys]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3In hydrogenchloride-ethyl acetate solution, remove Boc and generate the described compound of claim 1;
When the condensation product of step 8) is
Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3The time, will
Boc-Pro-Ala-Lys (Boc)-Lys[Boc-Pro-Ala-Lys (Boc)]-Asp[NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3In hydrogenchloride-ethyl acetate solution, remove Boc and generate the described compound of claim 1.
3. a pharmaceutical composition is characterized in that, this pharmaceutical composition contains the described compound of claim 1 that effective dose is gone up in treatment, and contains one or more pharmaceutically acceptable excipient or auxilliaryly add agent.
4. a pharmaceutical preparation is characterized in that, is the described compound of claim 1 and pharmaceutically acceptable excipient or the auxilliary mixture that adds agent are made tablet, capsule, pulvis, granule, lozenge or oral liquid.
5. the application of the described compound of claim 1 in the preparation thrombolytic agent.
6. the application of the described pharmaceutical composition of claim 3 in the preparation thrombolytic agent.
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| WO2019115812A1 (en) * | 2017-12-14 | 2019-06-20 | Universite De Strasbourg | Peptides for treatment and prevention of nonalcoholic fatty liver disease and fibrosis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1356338A (en) * | 2001-10-25 | 2002-07-03 | 北京明心源科技有限公司 | Separation, synthesis and application in medicine for P6A'S metabolic product |
| 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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2010
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1356338A (en) * | 2001-10-25 | 2002-07-03 | 北京明心源科技有限公司 | Separation, synthesis and application in medicine for P6A'S metabolic product |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| "基于脂质前药的自组装药物传递***";金义光 等;《中国医药工业杂志》;20051231;第36卷(第3期);第185-188页 * |
| 金义光 等."基于脂质前药的自组装药物传递***".《中国医药工业杂志》.2005,第36卷(第3期), |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019115812A1 (en) * | 2017-12-14 | 2019-06-20 | Universite De Strasbourg | Peptides for treatment and prevention of nonalcoholic fatty liver disease and fibrosis |
| IL275188B1 (en) * | 2017-12-14 | 2023-11-01 | Univ Strasbourg | Peptides for treatment and prevention of nonalcoholic fatty liver disease and fibrosis |
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