CN102477070A - Oligopeptide with thrombus dissolving activities and preparation method and application thereof - Google Patents
Oligopeptide with thrombus dissolving activities and preparation method and application thereof Download PDFInfo
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- CN102477070A CN102477070A CN2010105737904A CN201010573790A CN102477070A CN 102477070 A CN102477070 A CN 102477070A CN 2010105737904 A CN2010105737904 A CN 2010105737904A CN 201010573790 A CN201010573790 A CN 201010573790A CN 102477070 A CN102477070 A CN 102477070A
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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 4a-f in the specification, wherein in the formula, n is 6, 8, 10, 12, 14 or 16. Through animal experiments, the thrombus dissolving activities of the oligopeptide are evaluated, and the oligopeptide is proved to have excellent thrombus dissolving activities. The general formula 4a-f is shown in the specification.
Description
Technical field
The present invention has the oligopeptides of thrombus dissolving activity, belongs 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 through 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 through 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 oligopeptides, and estimated the thrombus dissolving activity of these oligopeptides, proved that oligopeptides of the present invention has outstanding thrombus dissolving activity through experimentation on animals with thrombus dissolving activity.
The present invention realizes through following technical scheme:
One of the object of the 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 preparation method of The compounds of this invention, it is characterized in that, comprises the steps:
1) Boc-Pro is Boc-Pro-Ala with the Ala condensation in anhydrous THF in the presence of NSC 57182 (DCC) and N-hydroxy-succinamide (HOSu);
2) in the presence of DCC and HOBt, Boc-Pro-Ala is Boc-Pro-Ala-Lys (Z)-OBzl or Boc-Pro-Ala-Lys-(Boc)-OBzl with Lys (Z)-OBzl or Lys-(Boc)-OBzl condensation in anhydrous THF;
3) in methyl alcohol with step 2) the product saponification of gained is Boc-Pro-Ala-Lys (Z) or Boc-Pro-Ala-Lys-(Boc);
4) in the presence of DCC, Boc-Asp is Boc-Asp [NHCH with the condensation of saturated fatty amine in anhydrous THF
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3, wherein, n is 6,8,10,12,14 or 16;
5) in 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) in the presence of DCC and HOBt, the step 3) products therefrom is Boc-Pro-Ala-Lys (Z)-Asp [NHCH with the condensation of step 5) products therefrom in anhydrous THF
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3Or Boc-Pro-Ala-Lys (Boc)-Asp [NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3
7) product when step 6) is Boc-Pro-Ala-Lys (Z)-Asp [NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3The time, at methyl alcohol, Pd/C and H
2Exist down,, obtain Boc-Pro-Ala-Lys-Asp [NHCH the product hydrogenolysis of step 6)
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3, in hydrogenchloride-ethyl acetate solution, deviate from Boc again, obtain The compounds of this invention;
When the product of step 6) is Boc-Pro-Ala-Lys (Boc)-Asp [NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3The time, in hydrogenchloride-ethyl acetate solution, the product of step 6) is deviate from Boc, obtain The compounds of this invention.
Three of the object of the 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 auxilliary and add agent.
Four of the object of the invention provides a kind of pharmaceutical prepn, and this pharmaceutical prepn is with The compounds of this invention and pharmaceutically acceptable excipient or assists the mixture that adds agent and process 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 the thrombus dissolving activity of oligopeptides of the present invention through experimentation on animals, has proved that oligopeptides of the present invention has outstanding thrombus dissolving activity.
Description of drawings
Fig. 1 is the general structure 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 4f.
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 are not to be understood that to be limitation of the present invention.
In an embodiment of the present invention, the used following implication of following breviary term:
The THF THF
HOBt N-hydroxy benzo triazole
The DCC NSC 57182
The TLC thin-layer chromatography
The Boc tertbutyloxycarbonyl
1.17g (5mmol) Boc-Asp is dissolved with the anhydrous THF of 20ml.In the solution that obtains, add 0.68g (5mmol) N-hydroxy benzo triazole (HOBT).After 10 minutes, under ice bath, add the solution of 2.06g (10mmol) NSC 57182 (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 NSC 30023 (DCU).Filtrate decompression concentrates, and removes THF.Residue is used the 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.ETHYLE ACETATE is used anhydrous Na mutually
2SO
4Drying, filtration, filtrate decompression are concentrated into dried, and residue obtains 1.64g (72%) target compound through column chromatography purification, is colourless powder.ESI-MS(m/e)：457[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 2.796g (12mmol) Boc-Asp and 4.145g (26.4mmol) CH
3(CH
2)
8CH
2NH
2Make 4.353g (71%) target compound, be the colorless solid 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 2g (8.6mmol) Boc-Asp and 3.493g (18.9mmol) CH
3(CH
2)
10CH
2NH
2Make 3.31g (68%) target compound, be the colorless solid 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 2.770 (11.9mmol) Boc-Asp and 5.572g (26.2mmol) CH
3(CH
2)
12CH
2NH
2Make 4.815g (65%) target compound, be the colorless solid 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..796g (12mmol) Boc-Asp and 6.362g (26.4mmol) CH
3(CH
2)
14CH
2NH
2Make 3.744g (46%) target compound, be the colorless solid 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 3.606g (49%) target compound, be the colorless solid 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 (5mmol) 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-Asp (NHC
8H
17)-NHC
8H
17Disappear, concentrating under reduced pressure is removed ETHYLE 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)：357[M+H]
+.
Embodiment 8 preparation Asp [NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3(2b)
Method 2.044g (4mmol) Boc-Asp [NHCH according to embodiment 7
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3Make 1.717g (84%) target compound, be the colorless solid 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 1.5g (2.65mmol) Boc-Asp [NHCH according to embodiment 7
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3Make 1.225g (92%) target compound, be the colorless solid 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 2.0g (3.21mmol) Boc-Asp [NHCH according to embodiment 7
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3Make 1.688g (94%) target compound, be the colorless solid powder.ESI-MS(m/e)：524[M+H]
+.
Method 2.716g (4mmol) Boc-Asp [NHCH according to embodiment 7
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3Make 2.240g (82%) target compound, be the colorless solid 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 (5mmol) Boc-Asp [NHCH according to embodiment 7
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3Make 2.86g (90%) target compound, be the colorless solid powder.ESI-MS(m/e)：636[M+H]
+.
Embodiment 13 preparation Boc-Pro-Ala
2.7g (12.6mmol) Boc-Pro with the anhydrous THF of 30ml, is added 1.59g (13.86mmol) N-hydroxy-succinamide (HOSu) in the solution that obtains.After 10 minutes, under the condition of ice bath, add 2.86g (13.86mmol) NSC 57182 (DCC) and the anhydrous THF of 20ml.At room temperature stir 10h, TLC (methylene chloride, 30: 1) shows that Boc-Pro disappears.Filtering NSC 30023 (DCU), THF is removed in decompression.Residue is used 250ml acetic acid ethyl dissolution, 5%NaHCO
3Give a baby a bath on the third day after its birth time, collect organic phase, decompression is removed ETHYLE ACETATE and is got white solid, THF dissolve reaction solution (I).1.23g (13.86mmol) Ala is dissolved in the 30ml water, obtains reaction solution (II).Under the ice bath reaction solution (II) is added in the reaction solution (I) saturated NaHCO
3Transfer pH to 8, at room temperature stir 16h, TLC (methylene chloride, 30: 1) shows that the condensation product of Boc-Pro and HOSu disappears.Saturated KHSO
4Transfer pH to 7, decompression removes THF, and residue is used saturated KHSO
4Transfer pH to 2.The solution that obtains is collected organic phase with ETHYLE ACETATE repeatedly extraction on a small quantity, and the saturated NaCl aqueous solution is given a baby a bath on the third day after its birth inferior.Organic phase is used anhydrous Na
2SO
4Drying, filtration, filtrate decompression are concentrated into dried, obtain 3.44g (95%) target compound, are the white solid powder.ESI-MS(m/e)：287[M+H]
+.
Embodiment 14 preparation Boc-Pro-Ala-Lys (Z)-OBzl
By 1.37g (4.8mmol) Boc-Pro-Ala and 2.17g (4mmol) Lys (Z)-OBzl, make 1.56g (61%) target compound according to the method for embodiment 1, be the colorless solid powder through column chromatography.ESI-MS(m/e)：639[M+H]
+.
Embodiment 15 preparation Boc-Pro-Ala-Lys (Boc)-OBzl
By 3.146g (11mmol) Boc-Pro-Ala and 2.966g (10mmol) Lys (Boc)-OBzl, make 4.472g (85%) target compound according to the method for embodiment 1, be the colorless solid powder through column chromatography.ESI-MS(m/e)：529[M+H]
+.
Embodiment 16 preparation Boc-Pro-Ala-Lys (Z)
1.92g (3mmol) Boc-Pro-Ala-Lys (z)-OBzl is dissolved in the methyl alcohol, ice bath stirs down and transfers pH to 12 with 2N NaOH, and ice bath stirred 4 hours, TLC (methylene dichloride: methyl alcohol=30: 1) get Boc-Pro-Ala-Lys (Z)-OBzl and disappear.Saturated KHSO
4Transfer pH to 6, decompression removes methyl alcohol.Saturated KHSO
4Transfer pH to 2, ETHYLE ACETATE is repeatedly extraction on a small quantity, collects organic phase, and the saturated NaCl aqueous solution is given a baby a bath on the third day after its birth inferior.Organic phase is used anhydrous Na
2SO
4Drying, filtration, filtrate decompression are concentrated into dried, make 1.48g (90%) target compound, are the colorless solid powder.ESI-MS(m/e)：547[M-H]
-.
Embodiment 17 preparation Boc-Pro-Ala-Lys (Boc)
Method according to embodiment 16 makes 2.397g (93%) target compound by 2.640g (mmol) Boc-Pro-Ala-Lys (Boc)-OBzl, is the white solid powder.ESI-MS(m/e)：515[M+H]
-.
Embodiment 18 preparation Boc-Pro-Ala-Lys (Z)-Asp [NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3(3a)
According to the method for embodiment 1 by 0.789g (1.44mmol) Boc-Pro-Ala-Lys (Z) and 0.465g (1.2mmol) Asp [NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3, make 0.659g (63%) target compound through column chromatography, be colourless powder.116 ℃ of Mp; [α]
D 25=-34.0 (c=0.5, methyl alcohol); ESI-MS (m/e): 872 [M+H]
+IR (KBr): 3324,3082,3937,2854,1690,1641,1538,1449,1398,1310,1247,1168,1089,1049,980,894,739,695,648.
1H NMR (300MHz, CDCl
3): δ/ppm=7.69-7.54 (m, 1H), 7.54-7.32 (m, 6H), 7.27-6.96 (m, 1H), 6.5-6.30 (m; 1H), 5.49-5.23 (m, 1H), 5.23-4.96 (m, 2H), 4.70 (s, 1H); 4.39-4.02 (m, 3H), 3.61-3.33 (m, 2H), 3.33-3.07 (m, 6H), 2.87-2.63 (m; 1H), 2.32-1.01 (m, 50H), 0.86 (t, J=6.6Hz, 6H).
Embodiment 19 preparation Boc-Pro-Ala-Lys (Boc)-Asp [NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3(3b)
According to the method for embodiment 1 by 1.096g (2mmol) Boc-Pro-Ala-Lys (Boc) and 0.986g (2.2mmol) Asp [NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3Make 0.567g (31%) target compound, be colourless powder.109 ℃ of Mp; [α]
D 25=-47.3 (c=0.56, methyl alcohol); ESI-MS (m/e): 942 [M+H]
+IR (KBr): 3292,3080,2927,2857,1696,1641,1547,1454,1401,1260,1164,1123,1025,1479,702.
1H NMR (300MHz, DMSO-d
6): δ/ppm=8.15-7.89 (m, 3H), 7.88-7.79 (m, 1H), 7.54-7.42 (m, 1H), 7.41-7.18 (m, 5H), 4.99 (s; 1H), and 4.55-4.38 (m, 1H), 4.38-4.19 (m, 1H), 4.17-3.03 (m, 2H), 3.09-2.89 (m, 6H); 2.12-2.03 (m, 2H), 1.88-1.72 (m, 2H), 1.72-1.57 (m, 2H), 1.55-1.48 (m, 2H); 1.45-1.28 (m, 16H), 1.28-1.09 (m, 32H), 0.86 (t, J=4.8Hz, J=6.6Hz, 6H).
Embodiment 20 preparation Boc-Pro-Ala-Lys (Z)-Asp [NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3(3c)
According to the method for embodiment 1 by 1.458g (2.2mmol) Boc-Pro-Ala-Lys (Z) and 1.007g (2mmol) Asp [NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3Make 857mg (45%) target compound, be colourless powder.IR(KBr)：3289，3088，2925，2859，19601641，1547，1455.51，1400，1304，1260，1164，1121，1025，980，918，767，696，579.ESI-MS(m/e)：1020[M+Na]
+.
1H?NMR(300MHz，DMSO-d
6)：δ/ppm＝8.07-7.98(m，3H)，7.83(s，1H)，7.45(s，1H)，7.34(s，5H)，7.18(s，1H)，5.0(s，2H)，4.49-4.36(m，2H)，4.10(s，2H)，3.01-2.95(m，7H)，2.74(s，1H)，2.33(s，1H)，2.07(s，1H)，1.87-1.77(m，4H)，1.69-1.65(m，2H)，1.65-1.55(m，2H)，1.39(s，6H)，1.32(s，11H)，1.23(s，40)，0.87-0.83(t，J＝6.3HzJ＝6.9Hz?3H).
Embodiment 21 preparation Boc-Pro-Ala-Lys (Z)-Asp [NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3(3d)
According to the method for embodiment 1 by 1.069g (1.95mmol) Boc-Pro-Ala-Lys (Z) and 0.785g (1.5mmol) Asp [NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3Make 0.664g (42%) target compound, be colourless powder.ESI-MS(m/e)：1055[M+H]
+.
1H?NMR(300MHz，DMSO-d
6)：δ/ppm＝8.05-7.94(m，3H)，7.85(s，1H)，7.46(s，1H)，7.34(s，5H)，7.20(s，1H)，5.00(s，2H)，4.41(s，1H)，4.10-4.00(m，4H)，3.06-2.96(m?8H)，2.73(s，1H)，2.28(s，1H)，2.06(s，1H)，1.99(s，1H)，1.76(s，4H)，1.64(s，2H)，1.52(s，2H)，1.38(s，6H)，1.32(s，13H)，1.23(s，44H)，0.87-0.83(t，J＝6.3Hz?J＝6.3Hz?3H).
Embodiment 22 preparation Boc-Pro-Ala-Lys (Boc)-Asp [NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3(3e)
According to the method for embodiment 1 by 0.514g (1mmol) Boc-Pro-Ala-Lys (Boc) and 0.677g (1.1mmol) Asp [NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3Make 370mg (34%) target compound, be colourless powder.99 ℃ of Mp; [α]
D 25=-42.5 (c=0.65, methyl alcohol); ESI-MS (m/e): 1075 [M+H]
+1098 [M+Na]
+, IR (KBr): 3289,3088,2923,2853,1690,1642,1544,1462,1397,1364,1456,1249,1170,718.
1H NMR (300MHz, DMSO-d
6): δ/ppm=8.09-7.82 (m, 4H), 6.22-6.05 (m, 2H), 4.92-4.19 (m, 4H), 3.37-3.09 (m, 10H), 2.18-1.62 (m, 8H), 1.62-1.1.18 (m, 68H), 0.89-0.84 (m, 6H).
Embodiment 23 preparation Boc-Pro-Ala-Lys (Z)-Asp [NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3(3f)
According to the method for embodiment 1 by 0.658g (1.2mmol) Boc-Pro-Ala-Lys (Z) and 0.635g (1mmol) Asp [NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3Make 0.606g (52%) target compound, be colourless powder.110 ℃ of Mp; [α]
D 25=-31.8 (c=0.6, methyl alcohol); ESI-MS (m/e): 1166 [M+H]
+IR (KBr): 3294,3073,2922,2853,1693,1642,1544,1456,1400,1257,1257,1165,1123,1021,697.
1H NMR (300MHz, CDCl
3): δ/ppm=7.46-7.31 (m, 5H), 6.76-6.59 (m, 1H), 5.11 (s, 2H), 4.78-4.68 (m, 1H), 4.50-4.09 (m; 3H), and 3.81-3.59 (m, 1H), 3.59-3.37 (m, 3H), 3.29-3.11 (m, 5H), 2.89-2.72 (m, 1H); 2.60-2.31 (m, 1H), 2.05 (s, 3H), 2.0-1.78 (m, 5H), 1.78-1.63 (m, 3H), 1.57-1.43 (m; 14H), 1.43-1.38 (m, 5H), 1.38-1.20 (m, 56H), 0.89 (t, J=4.8Hz, J=6.6Hz, 6H).
Embodiment 24 preparation Boc-Pro-Ala-Lys-Asp [NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3
With 0.871g (1mmol) Boc-Pro-Ala-Lys (Z)-Asp [NHCH
2(CH
2)
6CH
3]-NHCH
2-(CH
2)
6CH
3Use the 25ml dissolve with methanol, in solution, add 0.15g Pd/C, logical H
2(0.02Mba) stirring at room is to 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 0.691g (92%) target compound, are colourless powder.ESI-MS(m/e)：752[M+H]
+.
Embodiment 25 preparation Boc-Pro-Ala-Lys-Asp [NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3
According to the method for embodiment 24 by 0.997g (1mmol) Boc-Pro-Ala-Lys (Z)-Asp [NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3Make 0.804 (93%) target compound, be colourless powder.ESI-MS(m/e)：865[M+H]
+.
Embodiment 26 preparation Boc-Pro-Ala-Lys-Asp [NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3
According to the method for embodiment 24 by 0.954g (1mmol) Boc-Pro-Ala-Lys (Z)-Asp [NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3Make 0.747g (91%) target compound, be colourless powder.ESI-MS(m/e)：921[M+H]
+.
Embodiment 27 preparation Boc-Pro-Ala-Lys-Asp [NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3
According to the method for embodiment 24 by 1.165g (1mmol) Boc-Pro-Ala-Lys (Z)-Asp [NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3, make 0.928g (90%) target compound, be colourless powder.ESI-MS(m/e)：1032[M+H]
+.
Embodiment 28 preparation Pro-Ala-Lys-Asp [NHCH
2(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3(4a)
According to the method 0.376g (0.5mmol) of embodiment 7 by Boc-Pro-Ala-Lys-Asp [NHCH
2-(CH
2)
6CH
3]-NHCH
2(CH
2)
6CH
3Make 0.293g (90%) target compound, be colourless powder.Mp93 ℃; [α]
D 25=-45.8 (c=0.70, methyl alcohol); ESI-MS (m/e): 653 [M+H]
+IR (KBr): 3291,3060,2927,2857,1644,1547,1456,1379,1249,1156,710;
1H NMR (300MHz, DMSO): δ/ppm=8.80-8.74 (d, J=6.9Hz, 1H), 8.27-8.21 (d, J=7.2Hz, 1H); 8.10-8.04 (d, J=7.8Hz, 1H), 7.95-7.84 (m, 4H), 7.65-7.58 (s, 1H); 4.50-4.31 (m, 2h), 4.25-4.10 (m, 2H), 3.28-3.11 (m, 3H), 3.10-2.89 (m; 4H), and 2.79-2.65 (m, 2H), 2.35-2.26 (m, 1H), 1.97-1.79 (m, 3H); 1.62-1.47 (m, 4H), 1.46-1.11 (m, 29H), 0.79-0.91 (t, J=5.7Hz, 6H).
Embodiment 29 preparation Pro-Ala-Lys-Asp [NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
3(4b)
According to the method for embodiment 7 by Boc-Pro-Ala-Lys (Boc)-Asp [NHCH
2(CH
2)
8CH
3]-NHCH
2(CH
2)
8CH
30.150g make 109g (79%), target compound is colourless powder.105 ℃ of Mp; [α]
D 25=-51.8 (c=0.75, methyl alcohol); ESI-MS (m/e): 708 [M+H]
+IR (KBr): 3289,3073,2924,2855,1642,1547,1457,1382,1238,1164,1053,712,
1H-NMR (300MHz, DMSO-d
6): δ/ppm=8.67-8.57 (m, 1H), 8.38-8.17 (m, 2H), 8.02-7.94 (m, 1H), 7.64-7.56 (m; 1H), 4.49-4.30 (m, 3H), 3.09-2.93 (m, 6H), 2.73 (s, 2H); 2.29 (s, 1H), 2.21-2.10 (m, 1H), 1.87-1.44 (m, 14H); 1.44-0.97 (m, 42H), 0.85 (t, J=5.1Hz, J=6.9Hz, 6H).
Embodiment 30 preparation Pro-Ala-Lys-Asp [NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10CH
3(4c)
According to the method for embodiment 7 by 0.432g (0.5mmol) Boc-Pro-Ala-Lys-Asp [NHCH
2(CH
2)
10CH
3]-NHCH
2(CH
2)
10Make 0.336g (88%) target compound, be colourless powder.[α]
D 25=-33.43 (c=saves .57, methyl alcohol); IR (KBr): 3290,2922,2853,1643,1546,1463,1378,1294,1221,1160,714.
1H NMR (300MHz, DMSO-d
6): δ/ppm=8.79-8.77 (d, J=6.9Hz, 1H), 8.26-8.24 (d, J=7.2Hz, 1H), 8.10-7.95 (m, 2H); 7.61 (m, 3H), 4.74-4.34 (m, 2H), 4.20-4.13 (m, 2H), 3.26-3.17 (m, 2H); 3.10-2.90 (m, 2H), 2.46-2.28 (m, 3H), 1.87 (s, 3H), 1.68-1.45 (m, 5H); 1.4-1.28 (m, 11H), 1.25 (s, 36H), 0.87-0.83 (t, J=5.4Hz, 6H).
Embodiment 31 preparation Pro-Ala-Lys-Asp [NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3(4d)
According to the method for embodiment 7 by 185mg (0.18mmol) Boc-Pro-Ala-Lys-Asp [NHCH
2(CH
2)
12CH
3]-NHCH
2(CH
2)
12CH
3Make 152mg (94%) target compound, be colourless powder.109 ℃ of Mp; [α]
D 25=-58 (c=0.50, chloroforms); ESI-MS (m/e): 822 [M+H]
+IR (KBr): 3944,3433,3298,3229,2922,2855,2749,1742,1647,1545,1462,1387,1344,1292,1221,1169,1067,1003,723.
1H NMR (300MHz, DMSO-d
6): δ/ppm=10.07 (s, 1H), 8.39 (d, J=7.8Hz, 1H), 8.12 (d, J=7.8Hz, 1H); 4.66-4.62 (m, 1H), 4.36-4.31 (m, 1H), 4.26-4.18 (m, 2H), 4.02-3.99 (m, 4H); 3.24-3.17 (m, 2H), 2.74-2.72 (m, 4H), 2.34-2.29 (m, 1H), 1.91-1.85 (m, 3H); 1.68-1.52 (m, 9H), 1.27-1.23 (m, 49H), 0.86 (t, J=5.7Hz, J=6.6Hz, 6H).
Embodiment 32 preparation Pro-Ala-Lys-Asp [NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3(4e)
According to 7 the method implemented by 0.100g (0.09mmol) Boc-Pro-Ala-Lys (Boc)-Asp [NHCH
2(CH
2)
14CH
3]-NHCH
2(CH
2)
14CH
3Make 0.078g (81%) target compound, be colourless powder.104 ℃ of Mp; [α]
D 25=-19.7 (c=0.50, methyl alcohol); ESI-MS (m/e): 877 [M+H]
+IR (KBr): 3489,3293,3069,2921,2852,1644,1551,1465,1372,1303,1229,1146,1075,718,510.
1H NMR (300MHz, DMSO-d
6): δ/ppm=8.07-7.82 (m, 4H), 8.07-7.82 (m, 4H), 5.68-5.56 (m, 3H); 4.56-4.12 (m, 3H), 4.56-4.12 (m, 3H), 2.88-2.65 (m, 5H); 1.96-1.82 (m, 6H), 1.82-1.68 (m, 7H), 1.68-1.57 (m, 8H); 1.57-1.43 (m, 8H), 1.43-1.13 (m, 57H), 0.86 (m, 6H).
Embodiment 33 preparation Pro-Ala-Lys-Asp [NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3(4f)
According to the method for embodiment 7 by 0.52g (0.5mmol) Boc-Pro-Ala-Lys-Asp [NHCH
2(CH
2)
16CH
3]-NHCH
2(CH
2)
16CH
3Make 0.42g (89%) target compound, be colourless powder.97 ℃ of Mp; [α]
D 25=-11.4 (c=0.56, methyl alcohol); ESI-MS (m/e): 933 [M+H]
+IR (KBr): 3289,3068,2919,2851,1644,1546,1463,1376,1244,1164,714; 1H-NMR (300MHz, DMSO-d
6): δ/ppm=8.32-7.73 (m, 5H), 7.41-7.23 (m, 1H), 4.40-3.98 (m, 4H); 3.70-3.49 (m, 3H), 3.27-3.14 (m, 6H), 2.74 (s, 3H); 2.35-2.20 (m, 2H), 2.03-1.42 (m, 19H), 1.41-1.02 (m; 59H), 0.86-0.84 (t, J=5.1Hz, J=7.2Hz, 6H).
The thrombus dissolving activity of experimental example 14a-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 RCCA, 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 uses the Stainless Steel Wire coiled of diameter as 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 a polyethylene rubber tube, long 60mm, internal diameter 3.5mm; Two ends are identical polyethylene tube, long 100mm, internal diameter 1mm; External diameter 2mm, an end of this pipe pull into point pipe (being used to insert 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 to insert in the polyethylene rubber tube in stage casing).The equal silylanization 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.It is subsequent use to fill with heparin-saline solution (50IU/kg) in will managing through sharp pipe end with syringe.
The left external jugular vein of isolated from rat; Proximal part and distal end penetrate surgical thread respectively; On the left external jugular vein that exposes, cut an angle carefully; The point pipe of the bypass duct that above-prepared is good inserts 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 through the point pipe of the other end, this moment, syringe was not withdrawn polyethylene tube, clamped the flexible pipe between syringe and the polyethylene tube with mosquito forceps.Proximal part at RCCA stops blooding with bulldog clamp, RCCA 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 saline water (3ml/kg); Thrust away from the fixing nearly vein place of spiral of thrombus in the stage casing of the physiological salt soln of physiological salt soln of urokinase (20000IU/kg) or different concns The compounds of this invention through bypass tube (containing fixedly spiral of the thrombus of accurately weighing), opens bulldog clamp; Make blood flow flow to vein from artery through bypass duct; This is a rat arteriovenous shut Thrombolysis Model, slowly the liquid in the syringe is injected into (about 6min) in the blood, makes saline water (blank); Urokinase (positive control) or compound of the present invention are through blood circulation, and the sequential action of pressing vein-heart-artery is to thrombus.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 sees table 1.
Table 1 1nmol/kg 4a-f is to the influence of rat suppository loss of weight
a
A) n=10, wet weight of thrombus is represented with mean SDmg; B) compare p＜0.01 with the saline water group.
Experimental example 2 dosage are to the influence of thrombus dissolving activity in the 4f body
According to the experimental technique of experimental example 1, choose the best 4f of thrombolysis effect and investigate the thrombolysis activity under 1nmol/kg, 0.1nmol/kg and three dosage of 0.01nmol/kg.The result is as shown in table 2.The result shows, the thrombolytic effect show dose dependency of 4f.
Table 2
Table 2 is the influence of the dosage of 4f to the rat suppository loss of weight
a, wherein, a representes sample number, i.e. the number of used rat, and a) n=10, the thrombus loss of weight is represented with mean SD mg; B) with saline water and 0.1nmol/kg 4f group ratio, p＜0.01; C) with saline water and 0.01nmol/kg 4f group ratio, p＜0.05; D) compare p＜0.01 with saline water.
The particle diameter of the nanometer ball of 4a-f in experimental example 2 aqueous solution
1) particle diameter of 4a-f nanometer ball in the aqueous solution
4a-f is 10 when on the laser light scattering particle size analyzer, having 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 4a-f can be assembled into nanometer ball in the aqueous solution, and particle diameter is 166 to 554nm in the time of 25 ℃, and particle diameter is 141 to 291nm in the time of 37 ℃.
Table 3
2) form of the nanometer ball of 4a-f
4a-f is made into the aqueous solution that concentration is 1 * 10-12mg/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.With 4f is example, and the transmission electron microscope photo of 4f is as shown in Figure 3.Can know the nanometer ball of 4a-f formation rule from Fig. 3.
Claims (6)
1. the compound of following structure,
Wherein, n is 6,8,10,12,14 or 16.
2. a method for preparing the said compound of claim 1 is characterized in that, comprises the steps:
1) Boc-Pro is Boc-Pro-Ala with the Ala condensation in anhydrous THF in the presence of NSC 57182 (DCC) and N-hydroxy-succinamide (HOSu);
2) in the presence of DCC and HOBt, Boc-Pro-Ala is Boc-Pro-Ala-Lys (Z)-OBzl or Boc-Pro-Ala-Lys-(Boc)-OBzl with Lys (Z)-OBzl or Lys-(Boc)-OBzl condensation in anhydrous THF;
3) in methyl alcohol with step 2) the product saponification of gained is Boc-Pro-Ala-Lys (Z) or Boc-Pro-Ala-Lys-(Boc);
4) in the presence of DCC, Boc-Asp is Boc-Asp [NHCH with the condensation of saturated fatty amine in anhydrous THF
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3, wherein, n is 6,8,10,12,14 or 16;
5) in 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) in the presence of DCC and HOBt, the step 3) products therefrom is Boc-Pro-Ala-Lys (Z)-Asp [NHCH with the condensation of step 5) products therefrom in anhydrous THF
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3Or Boc-Pro-Ala-Lys (Boc)-Asp [NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3
7) product when step 6) is Boc-Pro-Ala-Lys (Z)-Asp [NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3The time, in the presence of methyl alcohol, Pd/C and H2,, obtain Boc-Pro-Ala-Lys-Asp [NHCH with the product hydrogenolysis of step 6)
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3, in hydrogenchloride-ethyl acetate solution, remove Boc again, obtain the said compound of claim 1;
When the product of step 6) is Boc-Pro-Ala-Lys (Boc)-Asp [NHCH
2(CH
2) nCH
3]-NHCH
2(CH
2) nCH
3The time, in hydrogenchloride-ethyl acetate solution, the product of step 6) is deviate from Boc, obtain the said compound of claim 1.
3. a pharmaceutical composition is characterized in that, contains the said compound of claim 1 that effective dose is gone up in treatment, and contains one or more pharmaceutically acceptable excipient or auxilliary and add agent.
4. a pharmaceutical prepn is characterized in that, is said compound of claim 1 and the perhaps auxilliary mixture that adds agent of pharmaceutically acceptable excipient are processed tablet, capsule, pulvis, granule, lozenge or oral liquid.
5. the application of the said compound of claim 1 in the preparation thrombolytic agent.
6. the application of the said compound of claim 3 in the preparation thrombolytic agent.
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| CN101134774A (en) * | 2006-08-31 | 2008-03-05 | 兰州大学 | Combined chemical modified endomorphin-1 and method for preparing same |
| 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 |
| CN101318992A (en) * | 2007-06-04 | 2008-12-10 | 北京大学 | Complete-basic amino acid oligopeptide, copper complex, synthesis method, self-assembly and application thereof |
| CN101538312A (en) * | 2009-05-08 | 2009-09-23 | 首都医科大学 | Preparation and applications of RGD-fatty amine series compound as tumor targeting vector material |
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| CN101134774A (en) * | 2006-08-31 | 2008-03-05 | 兰州大学 | Combined chemical modified endomorphin-1 and method for preparing same |
| 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 |
| CN101318992A (en) * | 2007-06-04 | 2008-12-10 | 北京大学 | Complete-basic amino acid oligopeptide, copper complex, synthesis method, self-assembly and application thereof |
| CN101538312A (en) * | 2009-05-08 | 2009-09-23 | 首都医科大学 | Preparation and applications of RGD-fatty amine series compound as tumor targeting vector material |
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