CN102477076B - Oligopeptide compound for thrombolysis, its preparation method and application - Google Patents

Oligopeptide compound for thrombolysis, its preparation method and application Download PDF

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CN102477076B
CN102477076B CN201010573793.8A CN201010573793A CN102477076B CN 102477076 B CN102477076 B CN 102477076B CN 201010573793 A CN201010573793 A CN 201010573793A CN 102477076 B CN102477076 B CN 102477076B
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obzl
boc
asp
pro
ala
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CN102477076A (en
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彭师奇
赵明
李珊
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Capital Medical University
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Abstract

The invention relates to an oligopeptide compound for thrombolysis, its preparation method and application. The oligopeptide compound for thrombolysis is as shown in general formula 6a-f (in the formula, n=6, 8, 10, 12, 14 and 16). In the invention, by means of animal experiments, the thrombolysis activity and targeting performance of the oligopeptide compound used for thrombolysis are evaluated. And in addition to excellent thrombolysis activity, the oligopeptide compound for thrombolysis in the invention is also proved to have targeting performance, can prevent new thrombus formation and have self-assembling performance. A general formula 6a-f is shown as below.

Description

Be used for oligopeptide compounds of thrombus dissolving and its preparation method and application
Technical field
The present invention relates to oligopeptide compounds having thrombus dissolving activity and its preparation method and application, relate in particular to the thrombus dissolving oligopeptides as shown in general formula 6a-f, belong 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.By the structural modification of PAK, being delayed vivo degradation speed and improved thrombus dissolving activity, is the important channel of oligopeptides thrombolytic agent research.
The activation of platelet membrane glycoprotein GPIIb/IIIa acceptor is the final shared pathway of platelet aggregation, in thrombosis, plays an important role.RGD sequence, as the ligands specific of activation GPIIb/IIIa acceptor, has antiplatelet aggregative activity and targeting thrombus.Utilize RGD sequence to prepare target thrombolytic drug to the targeting of thrombus position activated blood platelet, can reduce system fibrinolytic and hemorrhage complication, suppress thrombus position platelet aggregation simultaneously.
According to general understanding, containing the amphipathic molecule of polypeptide,, can there is self-assembly by noncovalent intermolecular interactions in the polypeptide that for example aliphatic alcohol chain is modified under suitable condition, forms nanostructure.By nanostructure, can improve conveying in vivo of polypeptide, delay degradation rate in vivo of polypeptide and improve the activity in vivo of polypeptide.
Summary of the invention
The object of the present invention is to provide a kind of oligopeptide compounds as shown in general formula 6a-f, and by experimentation on animals, evaluated the thrombus dissolving activity of these compounds.
The present invention is achieved through the following technical solutions these contents:
One of object of the present invention is to provide the compound of general formula 6a-f,
Figure BSA00000374951600021
general formula 6a-f
N=6,8,10,12,14 and 16 in formula.
Two of object of the present invention is to provide the preparation method of general formula 6a-f compound, comprises the steps:
(1) dicyclohexylcarbodiimide (DCC) there is lower Boc-Pro in anhydrous THF and and N-hydroxy-succinamide (HOSu) condensation be Boc-Pro-Osu, at NaHCO 3exist lower Boc-Pro-OSu to react with Ala and generate Boc-Pro-Ala;
(2) under DCC and HOBt existence, Boc-Pro-Ala is Boc-Pro-Ala-Lys (Z)-OBzl with Lys (Z)-OBzl condensation in anhydrous THF;
(3) under NaOH exists, in methyl alcohol, by Boc-Pro-Ala-Lys (Z)-OBzl saponification, be Boc-Pro-Ala-Lys (Z);
(4) under DCC exists, Boc-Asp (OBzl) is Boc-Asp (OBzl)-NHCH with the condensation of saturated fatty amine in anhydrous THF 2(CH 2) nCH 3; Described saturated fatty amine is C 8,10,12,14,16,18saturated fatty amine;
(5) Boc-Asp (OBzl)-NHCH in hydrogenchloride-ethyl acetate solution 2(CH 2) nCH 3slough Boc and generate Asp (OBzl)-NH CH 2(CH 2) nCH 3;
(6) under DCC and HOBt exist Boc-Pro-Ala-Lys (Z) in anhydrous THF with Asp (OBzl)-NH-CH 2(CH 2) nCH 3condensation is Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) nCH 3;
(7) NaOH exist under in methyl alcohol by Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NH-CH 2(CH 2) nCH 3saponification is Boc-Pro-Ala-Lys (Z)-Asp-NH CH 2(CH 2) nCH 3;
(8) Boc-Arg (NO under DCC and HOBt existence 2) in anhydrous THF, be Boc-Arg (NO with Gly-OBzl condensation 2)-Gly-OBzl;
(9) NaOH exist under in methyl alcohol by Boc-Arg (NO 2)-Gly-OBzl saponification is Boc-Arg (NO 2)-Gly;
(10) under DCC and HOBt existence, Boc-Asp (OBzl) is Boc-Asp (OBzl)-Ser (Bzl)-OBzl with Ser (Bzl)-OBzl condensation in anhydrous THF;
(11) in hydrogenchloride-ethyl acetate solution, Boc-Asp (OBzl)-Ser (Bzl)-OBzl sloughs Boc and generates Asp (OBzl)-Ser (Bzl)-OBzl;
(12) Boc-Arg (NO under DCC and HOBt existence 2)-Gly is Boc-Arg (NO with Asp (OBzl)-Ser (Bzl)-OBzl condensation in anhydrous THF 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl;
(13) Boc-Arg (NO in hydrogenchloride-ethyl acetate solution 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl sloughs Boc and generates Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl;
(14) Boc-Pro-Ala-Lys (Z)-Asp-NHCH under DCC and HOBt existence 2(CH 2) nCH 3in anhydrous THF with Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl condensation is Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) nCH 3;
(15) Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO under trifluoromethanesulfonic acid and trifluoracetic acid existence 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) nCH 3slough protecting group and generate general formula 6a-f compound.
Three of object of the present invention is to provide a kind of pharmaceutical composition, the general formula 6a-f compound that this pharmaceutical composition contains the upper effective dose for the treatment of, and contain one or more pharmaceutically acceptable excipient or additional dose.
Four of object of the present invention is to provide a kind of pharmaceutical preparation, is the mixture of general formula 6a-f compound and pharmaceutically acceptable excipient or additional dose is made to tablet, capsule, pulvis, granule, lozenge or oral liquid.
General formula 6a-f compound of the present invention and pharmaceutical composition can be used for preparing antithrombotic medicine.
The present invention has evaluated the antithrombotic acitivity of general formula 6a-f compound of the present invention by experimentation on animals, proved general formula 6a-f compound of the present invention except having outstanding thrombus dissolving activity, also there is the effect that Targeting Performance and the new bolt of prevention form, and independently fill performance.
Accompanying drawing explanation
Fig. 1 is the structural formula of general formula 6a-f of the present invention;
Fig. 2 is the synthetic route chart of general formula 6a-f compound of the present invention;
Fig. 3 is the transmission electron microscope photo of compound 6d.
In Fig. 2, i) DCC, HOSu, NaHCO 3and Ala; Ii) DCC, HOBt, NMM; Iii) the NaOH aqueous solution; Iv) hydrogenchloride/ethyl acetate solution (4N); V) trifluoromethanesulfonic acid, trifluoracetic acid.
Embodiment
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative completely, and they are only used for the present invention to be specifically described, and not should be understood to limitation of the present invention.
The preparation of embodiment 1Boc-Pro-Ala
1.075g (5.0mmol) Boc-Pro is dissolved in to the anhydrous THF of 20mL, under ice bath, in solution, adds 0.637g (5mmol) N-hydroxy-succinamide (HOSu), and make to dissolve completely.Under ice bath, dicyclohexylcarbodiimide (DCC) 1.236g (6.0mmol) that is dissolved in a small amount of anhydrous THF is added in reaction solution.Stirring at room 7h, TLC (petrol ether/ethyl acetate, 3: 1) monitoring Boc-Pro disappears.Filtering dicyclohexylurea (DCU) (DCU), the concentrated THF that removes of filtrate decompression.Ethyl acetate for enriched material (EA) is dissolved, and uses successively saturated NaHCO 3the aqueous solution is washed, the saturated NaCl aqueous solution is washed, and then EA layer is evaporated to dryly, adds appropriate THF to dissolve.Add again the Ala 0.489g (5.5mmol) that has been dissolved in a small amount of water, use NaHCO 3solid adjusts pH to 8-9, normal-temperature reaction 12h, and concentrating under reduced pressure is removed THF, adds 5mL water dissolution, uses saturated KHSO 4the aqueous solution is adjusted pH to 2, with EA repeatedly extraction on a small quantity, merges EA layer, is washed till neutrality, anhydrous sodium sulfate drying with the saturated NaCl aqueous solution.Filter, filtrate decompression is concentrated into dry, obtains 1.35g (94%) title compound, is white solid.ESI-MS(m/e)：285[M-H] -。
The preparation of embodiment 2Boc-Pro-Ala-Lys (Z)-OBzl
1.20g (4.2mmol) Boc-Pro-Ala is dissolved in to the anhydrous THF of 20mL, under ice bath, in solution, adds 0.54g (4.0mmol) HOBt, and make it to dissolve completely.Under ice bath, add 1.03g (5.0mmol) DCC.Obtain reaction solution I, stir 30 minutes.2.17g (4.0mmol) Lys (Z)-OBzl is suspended in the anhydrous THF of 20mL, then adds 1mlN-methylmorpholine (NMM), adjust pH 8-9, obtain reaction solution II.Reaction solution II is added in reaction solution I, under first ice bath, stir 1h, then stirring at room 4h, TLC (chloroform/methanol, 15: 1) monitoring Lys (Z)-OBzl disappears.Filtering DCU, the concentrated THF that removes of filtrate decompression.Residue 50mL acetic acid ethyl dissolution.The solution obtaining 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 layer anhydrous Na 2SO 4dry, to filter, filtrate decompression is concentrated into dry, obtains 1.92g (75%) title compound, is colourless powder.ESI-MS(m/e)：639[M+H] +。
The preparation of embodiment 3Boc-Pro-Ala-Lys (Z)
1.59g (3.0mmol) Boc-Pro-Ala-Lys (Z)-OBzl is dissolved in to 15mL methyl alcohol.Under ice bath, NaOH for solution (2N) aqueous solution obtaining is adjusted to pH12 and stirred 2h, TLC (methylene chloride/methanol, 15: 1) monitoring Boc-Pro-Ala-Lys (Z)-OBzl disappears.Dilute hydrochloric acid for reaction mixture (2N) is adjusted pH7, and concentrating under reduced pressure is except methyl alcohol.Dilute hydrochloric acid for residue (2N) is adjusted pH2, is extracted with ethyl acetate 3 times.Combined ethyl acetate layer, is washed till neutrality, anhydrous Na with the saturated NaCl aqueous solution 2sO 4dry.Filter, filtrate decompression is concentrated into dry, obtains 1.40g (85%) title compound, is colorless solid.ESI-MS(m/e)：547[M-H] -。
Embodiment 4Boc-Asp (OBzl)-NHCH 2(CH 2) 6cH 3(1a) preparation
Press the preparation method of embodiment 2 by 1.32g (4.1mmol) Boc-Asp (OBzl) and 0.52g (4.0mmol) C 8h 17nH 2obtaining 1.68g (97%) title compound, is colorless solid.ESI-MS(m/e)：401[M+H] +。
Embodiment 5Asp (OBzl)-NHCH 2(CH 2) 6cH 3(2a) preparation
By 1.52g (3.5mmol) Boc-Asp (OBzl)-NHCH 2(CH 2) 6cH 3be dissolved in hydrogenchloride-ethyl acetate solution that 15mL hydrogen cloride concentration is 4M, ice bath stirs 2 hours, TLC (methylene chloride/methanol, 15/1) monitoring raw material point disappears, concentrating under reduced pressure is removed ethyl acetate, residue repeatedly adds a small amount of ether and carries out concentrating under reduced pressure to remove hydrogen chloride gas, finally obtains 1.24g (95%) title compound, is faint yellow solid.ESI-MS(m/e)：369[M+H] +。
Embodiment 6Boc-Asp (OBzl)-NHCH 2(CH 2) 8cH 3(1b) preparation
Press the preparation method of embodiment 2 by 1.32g (4.1mmol) Boc-Asp (OBzl) and 0.63g (4.0mmol) C 10h 21nH 2obtaining 1.79g (97%) title compound, is colorless solid.ESI-MS(m/e)：429[M+H] +。
Embodiment 7Asp (OBzl)-NHCH 2(CH 2) 8cH 3(2b) preparation
Press the preparation method of embodiment 5 by 1.62g (3.5mmol) Boc-Asp (OBzl)-NHCH 2(CH 2) 8cH 3obtaining 1.33g (95%) title compound, is faint yellow solid.ESI-MS(m/e)：397[M+H] +。
Embodiment 8Boc-Asp (OBzl)-NHCH 2(CH 2) 10cH 3(1c) preparation
Press the preparation method of embodiment 2 by 1.32g (4.1mmol) Boc-Asp (OBzl) and 0.74g (4.0mmol) C 12h 25nH 2obtaining 1.89g (97%) title compound, is colorless solid.ESI-MS(m/e)：457[M+H] +。
Embodiment 9Asp (OBzl)-NHCH 2(CH 2) 10cH 3(2c) preparation
Press the preparation method of embodiment 5 by 1.72g (3.5mmol) Boc-Asp (OBzl)-NHCH 2(CH 2) 10cH 3obtaining 1.40g (94%) title compound, is faint yellow solid.ESI-MS(m/e)：426[M+H] +。
Embodiment 10Boc-Asp (OBzl)-NHCH 2(CH 2) 12cH 3(1d) preparation
Press the preparation method of embodiment 2 by 1.32g (4.1mmol) Boc-Asp (OBzl) and 0.85g (4.0mmol) C 14h 29nH 2reaction, obtains 1.99g (96%) title compound, is colorless solid.ESI-MS(m/e)：485[M+H] +。
Embodiment 11Asp (OBzl)-NHCH 2(CH 2) 12cH 3(2d) preparation
Press the preparation method of embodiment 5 by 1.81g (3.5mmol) Boc-Asp (OBzl)-NHCH 2(CH 2) 12cH 3obtaining 1.51g (95%) title compound, is faint yellow solid.ESI-MS(m/e)：454[M+H] +。
Embodiment 12Boc-Asp (OBzl)-NHCH 2(CH 2) 14cH 3(1e) preparation
Press the preparation method of embodiment 2 by 1.32g (4.1mmol) Boc-Asp (OBzl) and 0.96g (4.0mmol) C 16h 33nH 2obtaining 2.09g (96%) title compound, is colorless solid.ESI-MS(m/e)：513[M+H] +.
Embodiment 13Asp (OBzl)-NHCH 2(CH 2) 14cH 3(2e) preparation
Press the preparation method of embodiment 5 by 1.91g (3.5mmol) Boc-Asp (OBzl)-NHCH 2(CH 2) 14cH 3obtaining 1.61g (96%) title compound, is faint yellow solid.ESI-MS(m/e)：482[M+H] +。
Embodiment 14Boc-Asp (OBzl)-NHCH 2(CH 2) 16cH 3(1f) preparation
Press the preparation method of embodiment 2 by 1.32g (4.1mmol) Boc-Asp (OBzl) and 1.08g (4.0mmol) C 18h 37nH 2obtaining 2.19g (96%) title compound, is colorless solid.ESI-MS(m/e)：541[M+H] +。
Embodiment 15Asp (OBzl)-NHCH 2(CH 2) 16cH 3(2f) preparation
Press the preparation method of embodiment 5 by 2.01g (3.5mmol) Boc-Asp (OBzl)-NHCH 2(CH 2) 16cH 3obtaining 1.69g (94%) title compound, is faint yellow solid.ESI-MS(m/e)：510[M+H] +。
Embodiment 16Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 6cH 3(3a) preparation
Press the preparation method of embodiment 2 by 1.10g (2.0mmol) Boc-Pro-Ala-Lys (Z) and 0.78g (2.1mmol) Asp (OBzl)-NHCH 2(CH 2) 6cH 3making 1.46g (85%) title compound, is colorless solid.ESI-MS(m/e)：865[M+H] +。
Embodiment 17Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 6cH 3(4a) preparation
Press the preparation method of embodiment 3 by 1.38g (1.6mmol) Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 6cH 3obtaining 1.05g (85%) title compound, is colourless powder.ESI-MS(m/e)：773[M-H] -。
Embodiment 18Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 8cH 3(3b) preparation
Press the preparation method of embodiment 2 by 1.10g (2.0mmol) Boc-Pro-Ala-Lys (Z) and 0.84g (2.1mmol) Asp (OBzl)-NHCH 2(CH 2) 8cH 3making 1.52g (85%) title compound, is colorless solid.ESI-MS(m/e)：893[M+H] +。
Embodiment 19Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 8cH 3(4b) preparation
Press the preparation method of embodiment 3 by 1.43g (1.6mmol) Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 8cH 3obtaining 1.08g (84%) title compound, is colourless powder.ESI-MS(m/e)：801[M-H] -。
Embodiment 20Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 10cH 3(3c) preparation
Press the preparation method of embodiment 2 by 1.10g (2.0mmol) Boc-Pro-Ala-Lys (Z) and 0.90g (2.1mmol) Asp (OBzl)-NHCH 2(CH 2) 10cH 3making 1.57g (85%) title compound, is colorless solid.ESI-MS(m/e)：921[M+H] +。
Embodiment 21Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 10cH 3(4c) preparation
Press the preparation method of embodiment 3 by 1.47g (1.6mmol) Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 10cH 3obtaining 1.14g (86%) title compound, is colourless powder.ESI-MS(m/e)：829[M-H] -。
Embodiment 22Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 12cH 3(3d) preparation
Press the preparation method of embodiment 2 by 1.10g (2.0mmol) Boc-Pro-Ala-Lys (Z) and 0.95g (2.1mmol) Asp (OBzl)-NHCH 2(CH 2) 12cH 3making 1.63g (86%) title compound, is colorless solid.ESI-MS(m/e)：949[M+H] +。
Embodiment 23Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 12cH 3(4d) preparation
Press the preparation method of embodiment 3 by 1.52g (1.6mmol) Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 12cH 3obtaining 1.15g (84%) title compound, is colourless powder.ESI-MS(m/e)：858[M-H] -。
Embodiment 24Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 14cH 3(3e) preparation
Press the preparation method of embodiment 2 by 1.10g (2.0mmol) Boc-Pro-Ala-Lys (Z) and 1.01g (2.1mmol) Asp (OBzl)-NHCH 2(CH 2) 14cH 3making 1.66g (85%) title compound, is colorless solid.ESI-MS(m/e)：977[M+H] +。
Embodiment 25Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 14cH 3(4e) preparation
Press the preparation method of embodiment 3 by 1.56g (1.6mmol) Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 14cH 3obtaining 1.22g (86%) title compound, is colourless powder.ESI-MS(m/e)：886[M-H] -。
Embodiment 26Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 16cH 3(3f) preparation
Press the preparation method of embodiment 2 by 1.10g (2.0mmol) Boc-Pro-Ala-Lys (Z) and 1.07g (2.1mmol) Asp (OBzl)-NHCH 2(CH 2) 16cH 3making 1.73g (86%) title compound, is colorless solid.ESI-MS(m/e)：1005[M+H] +。
Embodiment 27Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 16cH 3(4f) preparation
Press the preparation method of embodiment 3 by 1.61g (1.6mmol) Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) 16cH 3obtaining 1.24g (85%) title compound, is colourless powder.ESI-MS(m/e)：914[M-H] -。
Embodiment 28Boc-Arg (NO 2the preparation of)-Gly-OBzl
Press the preparation method of embodiment 2 by 1.60g (5.0mmol) Boc-Arg (NO 2) and 1.69g (5.0mmol) Gly-OBzl obtain 2.26g (97%) title compound, be faint yellow solid.ESI-MS(m/e)：466[M+H] +。
Embodiment 29Boc-Arg (NO 2the preparation of)-Gly
Press the preparation method of embodiment 3 by 1.63g (3.5mmol) Boc-Arg (NO 2)-Gly-OBzl obtains 1.24g (94%) title compound, is colorless solid.ESI-MS(m/e)：375[M-H] -。
The preparation of embodiment 30Boc-Asp (OBzl)-Ser (Bzl)-OBzl
By the preparation method of embodiment 2, by 1.62g (5.0mmol) Boc-Asp (OBzl) and 1.61g (5.0mmol) Ser (Bzl)-OBzl, obtaining 2.87g (97%) title compound, is faint yellow solid.ESI-MS(m/e)：592[M+H] +。
The preparation of embodiment 31Asp (OBzl)-Ser (Bzl)-OBzl
By the preparation method of embodiment 5, by 2.36g (4.0mmol) Boc-Asp (OBzl)-Ser (Bzl)-OBzl, obtaining 1.94g (97%) title compound, is faint yellow solid.ESI-MS(m/e)：500[M+H] +.
Embodiment 32Boc-Arg (NO 2the preparation of)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
Press the preparation method of embodiment 2 by 1.31g (3.5mmol) Boc-Arg (NO 2)-Gly and 1.75g (3.5mmol) Asp (OBzl)-Ser (Bzl)-OBzl obtains 2.08g (70%) title compound, is colorless solid.ESI-MS(m/e)：849[M+H] +。
Embodiment 33Arg (NO 2the preparation of)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
Press the preparation method of embodiment 5 by 1.87g (2.2mmol) Boc-Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl makes 1.56g (95%) title compound, is faint yellow solid.ESI-MS(m/e)：747[M+H] +。
Embodiment 34 prepares Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 6cH 3(5a)
Press the preparation method of embodiment 2 by 0.74g (1.0mmol) Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 6cH 3and 0.74g (1.0mmol) Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl obtains 1.01g (67%) title compound, is colorless solid.ESI-MS(m/e)：1529[M+Na] +；Mp：167-168℃； (c＝0.43，CH 3OH).IR(KBr)：3299.18，3065.65，2930.02，2858.81，1646.87，1539.29，1455.09，1405.18，1366.55，1259.97，1164.86，1027.88，737.32，697.89，609.68. 1H?NMR(300MHz，DMSO)：δ/ppm＝8.42(m，1H)，8.20-8.02(m，3H)，8.02-7.69(m，6H)，7.35-7.17(m，20H)，5.13-4.95(m，6H)，4.89-4.69(m，1H)，4.69-4.38(m，4H)，4.38-3.89(m，4H)，3.89-3.49(m，4H)，3.50-3.40(m，3H)，3.18-3.05(s，2H)，3.05-2.85(s，4H)，2.85-2.71(s，1H)，2.20-2.05(s，1H)，1.90-1.65(s，4H)，1.59-1.45(s，4H)，1.45-1.25(m，14H)，1.25-1.15(m，14H)，1.13-1.05(m，5H)，0.88-0.85(t，J＝5.7Hz，J＝6.6Hz，3H).
Embodiment 35 prepares Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 8cH 3(5b)
Press the preparation method of embodiment 2 by 0.74g (1.0mmol) Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 8cH 3and 0.74g (1.0mmol) Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl obtains 1.00g (65%) title compound, is colorless solid.ESI-MS(m/e)：1557[M+Na] +.Mp：167-168℃.
Figure BSA00000374951600101
(c＝0.20，CH 3OH).IR(KBr)：3299.29，2928.38，1646.93，1539.68，1455.22，1404.43，1366.30，1259.34，1164.20，736.38，697.38. 1H?NMR(300MHz，DMSO)：δ/ppm＝8.88-8.38(m，2H)，8.38-8.05(m，3H)，8.05-7.75(m，6H)，7.50-7.05(m，20H)，5.22-4.95(m，6H)，4.89-4.78(m，1H)，4.78-4.38(m，4H)，4.38-3.99(m，4H)，3.89-3.57(m，4H)，3.19-2.88(m，6H)，2.88-2.67(s，1H)，2.20-1.87(m，1H)，1.87-1.65(s，5H)，1.65-1.05(m，40H)，0.88-0.79(d，J＝6.6Hz，3H).
Embodiment 36 prepares Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 10cH 3(5c)
Press the preparation method of embodiment 2 by 0.74g (1.0mmol) Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 10cH 3and 0.74g (1.0mmol) Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl obtains 1.03g (66%) title compound, is colorless solid.ESI-MS(m/e)：1585[M+Na] +.Mp：168-169℃.
Figure BSA00000374951600102
(c＝0.27，CH 3OH).IR(KBr)：3301.47，2926.86，2855.08，1733.73，1646.86，1533.45，1455.40，1408.28，1366.89，1257.77，1172.34，738.73，698.19，677.18. 1H?NMR(300MHz，DMSO)：δ/ppm＝8.79-8.32(m，2H)，8.32-8.07(m，3H)，8.07-7.55(m，5H)，7.55-7.07(m，20H)，5.43-4.90(m，6H)，4.90-4.69(d，J＝5.1Hz，1H)，4.69-4.39(m，4H)，4.39-3.95(m，4H)，3.95-3.55(m，4H)，3.55-3.20(s，9H)，3.20-2.67(m，7H)，2.67-2.33(m，13H)，2.33-1.89(m，1H)，1.89-0.98(m，45H)，0.98-0.72(d，J＝5.1Hz，3H).
Embodiment 37 prepares Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 12cH 3(5d)
Press the preparation method of embodiment 2 by 0.74g (1.0mmol) Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 12cH 3and 0.74g (1.0mmol) Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl obtains 1.08g (68%) title compound, is colorless solid.ESI-MS(m/e)：1613[M+Na] +.Mp：167-168℃.
Figure BSA00000374951600111
(c＝0.27，CH 3OH).IR(KBr)：3299.14，2926.14，2854.15，1733.88，1646.98，1540.39，1455.44，1404.28，1365.99，1258.88，1164.82，697.11. 1H?NMR(300MHz，DMSO)：δ/ppm＝8.41-8.13(m，1H)，8.13-7.66(m，3H)，7.55-6.98(m，20H)，5.32-4.92(m，6H)，4.92-4.69(m，1H)，4.69-4.39(m，4H)，4.39-3.99(m，4H)，3.86-3.55(m，4H)，3.20-3.03(m，2H)，3.03-2.82(m，5H)，2.20-1.91(m，1H)，1.91-1.12(m，52H)，0.90-0.62(t，J＝5.7Hz，J＝4.8Hz，3H).
Embodiment 38 prepares Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 14cH 3(5e)
Press the preparation method of embodiment 2 by 0.74g (1.0mmol) Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 14cH 3and 0.74g (1.0mmol) Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl obtains 1.13g (70%) title compound, is colorless solid.ESI-MS(m/e)：1641[M+Na] +.Mp：169-171℃.
Figure BSA00000374951600112
(c＝0.30，CH 3OH).IR(KBr)：3299.35，2925.72，2854.04，1647.11，1539.57，1455.36，1404.42，1365.96，1258.21，1164.67，736.14，697.20. 1H?NMR(300MHz，DMSO)：δ/ppm＝8.62-8.38(m，2H)，8.38-8.15(m，3H)，8.15-7.60(m，6H)，7.55-7.13(m，20H)，5.40-4.95(m，6H)，4.95-4.72(m，1H)，4.72-4.39(m，4H)，4.39-3.95(m，4H)，3.95-3.50(m，4H)，3.21-3.07(s，2H)，3.07-2.88(s，4H)，2.88-2.70(d，J＝8.1Hz，1H)，2.70-2.39(m，6H)，2.22-1.92(d，J＝10.2Hz，1H)，1.92-0.99(m，56H)，0.99-0.69(t，J＝5.1Hz，J＝6.6Hz，3H).
Embodiment 39 prepares Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 16cH 3(5f)
Press the preparation method of embodiment 2 by 0.74g (1.0mmol) Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) 16cH 3and 0.74g (1.0mmol) Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl obtains 1.10g (67%) title compound, is colorless solid.ESI-MS(m/e)：1669[M+Na]+.Mp：170-171℃. (c＝0.25，CH 3OH).IR(KBr)：3299.51，2925.02，2853.42，1647.22，1540.18，1455.25，1404.26，1366.12，1259.07，1164.48，736.08，697.07.1H?NMR(300MHz，DMSO)：8.72-8.36(m，2H)，8.36-8.09(m，3H)，8.09-7.72(m，6H)，7.49-7.12(m，20H)，5.30-4.90(m，6H)，4.90-4.69(m，1H)，4.69-4.39(m，4H)，4.39-3.92(m，4H)，3.92-3.51(m，4H)，3.17-3.05(s，2H)，3.05-2.68(s，2H)，2.22-1.89(s，1H)，1.89-1.12(m，61H)，0.98-0.72(t，J＝6.0Hz，J＝6.6Hz，3H).
Embodiment 40 prepares Pro-Ala-Lys-Asp (Arg-Gly-Asp-Ser)-NHCH 2(CH 2) 6cH 3(6a)
By 100mg (0.07mmol) Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 6cH 3(5a) be placed in the eggplant bottle of 250mL with drying tube, under ice bath, add 4mL trifluoracetic acid and 1mL trifluoromethanesulfonic acid, reaction 1h, TLC demonstration reacts completely, and adds a large amount of anhydrous diethyl ethers, stirs 5min, standing, the supernatant liquor that inclines, in triplicate, drain, add a small amount of distilled water and dissolve, weak ammonia is adjusted pH7, by Sephadex post desalination, freeze-drying, obtains 35mg (67%) title compound, is colorless solid.ESI-MS(m/e)：955[M-H] -.Mp：135-137℃.
Figure BSA00000374951600122
(c＝0.25，CH 3OH).IR(KBr)：3365.03，2931.89，1653.46，1543.68，1403.33，621.90. 1H?NMR(300MHz，D 2O)：δ/ppm＝4.72-4.54(m，3H)，4.39-4.15(m，5H)，3.99-3.80(s，2H)，3.80-3.72(d，J＝4.5Hz，2H)，3.55-3.25(m，3H)，3.25-3.05(m，5H)，3.05-2.81(m，3H)，2.81-2.47(m，5H)，2.47-2.32(m，1H)，2.10-1.92(m，3H)，1.92-1.82(s，5H)，1.82-1.50(m，8H)，1.50-1.30(d，J＝5.1Hz，8H)，1.30-1.05(s，10H)，0.96-0.66(t，3H).
Embodiment 41 prepares Pro-Ala-Lys-Asp (Arg-Gly-Asp-Ser)-NHCH 2(CH 2) 8cH 3(6b)
Press the method for embodiment 40 by 100mg (0.065mmol) Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 8cH 3(5b) obtaining 33mg (62%) title compound, is colorless solid.ESI-MS(m/e)：983[M-H] -.Mp：137-138℃.
Figure BSA00000374951600123
(c＝0.50，CH 3OH).IR(KBr)：3301.36，2929.16，1652.40，1554.54，1405.70，654.10. 1H?NMR(300MHz，D 2O)：δ/ppm＝4.72-4.58(s，2H)，4.58-4.15(m，6H)，4.15-3.88(s，2H)，3.88-3.75(d，J＝3.9Hz，2H)，3.75-3.48(s，1H)，3.46-3.05(m，7H)，3.02-2.88(t，J＝7.2Hz，J＝7.5Hz，2H)，2.88-2.55(m，4H)，2.55-2.30(m，1H)，2.13-0.89(m，36H)，0.89-0.73(d，J＝6.3Hz，3H).
Embodiment 42 prepares Pro-Ala-Lys-Asp (Arg-Gly-Asp-Ser)-NHCH 2(CH 2) 10cH 3(6c)
Press preparation method in embodiment 40, by 100mg (0.06mmol) Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 10cH 3(5c) obtaining 38mg (64%) title compound, is colorless solid.ESI-MS(m/e)：1011[M-H] -.Mp：138-140℃.
Figure BSA00000374951600131
(c＝0.23，CH3OH).IR(KBr)：3284.18，2927.16，1653.98，1551.93，1402.11. 1H?NMR(300MHz，D 2O)：δ/ppm＝4.56-4.12(m，6H)，4.12-3.70(ss，5H)，3.70-3.55(m，1H)，3.55-3.25(ss，3H)，3.25-3.02(d，J＝20.4Hz，4H)，3.02-2.88(s，2H)，2.88-2.52(s，4H)，2.52-2.21(s，1H)，2.21-1.53(m，14H)，1.53-0.89(m，25H)，0.89-0.69(s，3H).
Embodiment 43 prepares Pro-Ala-Lys-Asp (Arg-Gly-Asp-Ser)-NHCH 2(CH 2) 12cH 3(6d)
Press preparation method in embodiment 40, by 100mg (0.055mmol) Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 12cH 3(5d) obtaining 39mg (67%) title compound, is colorless solid.ESI-MS(m/e)：1039[M-H] -.Mp：137-139℃.
Figure BSA00000374951600132
(c＝0.35，CH 3OH).IR(KBr)：3291.75，2926.21，2855.99，1653.35，152.66，1402.77. 1H?NMR(300MHz，D 2O)：δ/ppm＝4.69-4.52(s，2H)，4.52-4.15(m，5H)，4.15-3.66(s，2H)，3.80-3.72(ss，2H)，3.55-3.25(m，3H)，3.25-3.05(m，5H)，3.05-2.81(m，3H)，2.81-2.47(m，4H)，3.66-3.27(ss，2H)，3.27-3.02(m，4H)，3.02-2.88(m，3H)，2.88-2.51(s，4H)，2.51-2.08(s，2H)，2.08-1.52(m，15H)，1.52-0.88(m，29H)，0.88-0.72(s，3H).
Embodiment 44 prepares Pro-Ala-Lys-Asp (Arg-Gly-Asp-Ser)-NHCH 2(CH 2) 14cH 3(6e)
Press preparation method in embodiment 40, by 100mg (0.05mmol) Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 14cH 3(5e) obtaining 37mg (65%) title compound, is colorless solid.ESI-MS(m/e)：1067[M-H] -.Mp：138-139℃.
Figure BSA00000374951600141
(c＝0.20，CH 3OH).IR(KBr)：3323.92，2927.29，1656.64，1548.83，1255.62. 1H?NMR(300MHz，D 2O)：δ/ppm＝4.69-4.60(s，2H)，4.60-4.08(m，6H)，4.08-3.58(m，5H)，3.58-3.27(m，3H)，3.27-3.02(m，4H)，3.02-2.83(m，3H)，2.83-2.48(m，4H)，2.48-2.20(s，2H)，2.13-0.84(m，51H)，0.84-0.74(s，3H).
Embodiment 45 prepares Pro-Ala-Lys-Asp (Arg-Gly-Asp-Ser)-NHCH 2(CH 2) 16cH 3(6f)
Press preparation method in embodiment 40, by 100mg (0.045mmol) Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) 16cH 3(5f) obtaining 39mg (66%) title compound, is colorless solid.ESI-MS(m/e)：1095[M-H] -.Mp：139-141℃.
Figure BSA00000374951600142
(c＝0.10，CH 3OH).IR(KBr)：3286.03，2925.41，2854.99，1653.38，1549.37，1398.71.1H-NMR(300MHz，D 2O)：δ/ppm＝4.69-4.60(s，1H)，4.48-4.12(ss，5H)，4.12-3.58(s，4H)，3.49-2.85(m，10H)，2.85-2.25(ss，5H)，2.12-1.51(m，16H)，1.51-0.82(m，40H)，0.82-0.74(s，3H)。
Thrombus dissolving activity in the body of experimental example 16a-f
200-220g male SD rat is anaesthetized with 20% urethane solution (6ml/kg, i.p.).Anesthetized rat dorsal position is fixed, and separates right common carotid artery, in proximal part folder bulldog clamp, proximal part and distal end penetrate respectively surgical thread, the surgical thread of distal end are clamped with mosquito forceps in fur, in distal end intubate, unclamp bulldog clamp, emit about 1ml arterial blood and be contained in the EP pipe of 1ml.Past vertical fixing Glass tubing (long 15mm, internal diameter 2.5mm, external diameter 5.0mm, the pipe end, seals with plug) and the middle 0.1ml rat artery blood that injects, the rapid thrombus standing bolt that inserts a stainless steel material in past pipe.The fixing spiral of this thrombus is coiled into the Stainless Steel Wire that diameter is 0.2mm, the long 12mm of spiral part, and containing 15 bung flanges, the diameter of bung flange is 1.0mm, and holder handle is connected with spiral, and long 7.0mm, is question mark type.After blood coagulation 15min, open the plug of Glass tubing bottom, with the holder handle of the fixing spiral of the fixing thrombus of tweezers, from Glass tubing, take out the fixing spiral of the thrombus being wrapped up by thrombus, accurately weigh.
Bypass intubate forms by 3 sections, and 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 (for inserting rat carotid artery or vein), external diameter 1mm, the outer cover one segment length 7mm of the other end, the polyethylene tube (overstriking, for inserting in the polyethylene rubber tube in stage casing) of external diameter 3.5mm.The equal silanization of inwall of 3 sections of pipes.Fixing the thrombus of thrombus parcel spiral is put into stage casing polyethylene rubber tube, and the two ends of sebific duct are nested with two poly butt ends that add respectively.Standby by filling with heparin-saline solution (50IU/kg) in pipe by sharp pipe end with syringe.
Separate the left external jugular vein of rat, proximal part and distal end penetrate respectively surgical thread, on the left external jugular vein exposing, cut carefully an angle, the sharp pipe of the bypass duct preparing is inserted to the proximal part of left external jugular vein opening above by angle, away from the interior thrombus in bypass tube stage casing (containing the fixing spiral of the thrombus of accurately weighing), fix the holder handle of spiral simultaneously.The heparin-saline (50IU/kg) that pushes accurate amount with syringe by the sharp pipe of the other end, now syringe is not withdrawn polyethylene tube, clamps the flexible pipe between syringe and polyethylene tube with mosquito forceps.Proximal part in right common carotid artery stops blooding with bulldog clamp, from bulldog clamp, right common carotid artery is being cut to an angle carefully nearby.From the tip of polyethylene tube, extract syringe, the tip of polyethylene tube is inserted to the proximal part of artery angle.The two ends of bypass duct all use No. 4 sutures and arteriovenous to fix.
With scalp acupuncture by physiological saline (3ml/kg), the normal saline solution (20000IU/kg) of urokinase or the normal saline solution of 1nmol/kg 6a-f are by the stage casing (containing the fixing spiral of the thrombus of accurately weighing) of bypass tube, thrust the nearly vein place away from the fixing spiral of thrombus, open bulldog clamp, make blood flow from artery, flow to vein by bypass duct, this is rat arteriovenous shut Thrombolysis Model, slowly the liquid in syringe is injected into (about 6min) in blood, make physiological saline (blank), urokinase (positive control) or compound of the present invention are by blood circulation, press the sequential action of vein-heart-artery to thrombus.Timing during from start injection, after 1h, from bypass duct, removal of thromboses is fixed spiral, accurately weighs.Calculate thrombus in every rat bypass duct and fix the of poor quality of spiral administration front and back, thrombolysis activity in the body of statistics assessing compound.Result is as shown in table 4.Result shows, 6a-f has thrombolysis activity in outstanding body.
Table 1
Table 1 is the impact of 1nmol/kg 6a-f on rat suppository loss of weight a, wherein, a represents sample number, i.e. the number of rat used, and a) n=12, thrombus loss of weight represents with mean value ± SD mg; B) with physiological saline group ratio, p < 0.01.
The impact of experimental example 2 dosage on thrombus dissolving activity in 6d body
According to the experimental technique of experimental example 1, choose the best 6d of thrombolytic effect and investigate the thrombolysis activity under 10nmol/kg, 1nmol/kg and tri-dosage of 0.1nmol/kg.Result is as shown in table 2.Result shows, the thrombolytic effect show dose dependency of 6d.
Table 2
Figure BSA00000374951600161
Table 2 is the impact on rat suppository loss of weight of the dosage of 6d a, wherein, a represents sample number, i.e. the number of rat used, and a) n=10, thrombus loss of weight represents with mean value ± SD mg; B) with physiological saline and 1nmol/kg 6d group ratio, p < 0.05; C) with physiological saline and 0.1nmol/kg 6d group ratio, p < 0.01; D) compared with physiological saline, p < 0.01.
The external thrombus dissolving activity of experimental example 36a-f
1) making of thrombosis device
By internal diameter 4mm, external diameter 5.5mm, one section of Glass tubing of length 18mm is placed on a quick detachable base of plastics, and the seam crossing of Glass tubing and plastic feet seals with one section of emulsion tube.In Glass tubing, place a Stainless Steel Wire spiral, screw diameter 1mm, length 20mm, comprise the long hook of 2mm of one end, blood be set in stainless steel spiral around, while weighing, thrombus can be hung up, and when hatching, thrombus can be hung in the solution of reaction flask, do not encounter wall, in order to avoid damage thrombus.
2) making of reaction flask
With the 10ml cillin bottle with rubber plug, on rubber plug, wear a Stainless Steel Wire, one end in bottle curves hook, thrombus hangs on hook, be suspended in bottle interior testing compound solution, Stainless Steel Wire can move up and down on rubber plug, regulates the height of thrombus in solution, and it is just immersed in solution to be measured.The simulation of internal milieu: estimate that according to rat mean body weight every rat has 13ml blood, if rat thrombus in vivo model, blood that may about 8ml can touch thrombus, therefore add 8ml solution to be measured in reaction flask, hatches at 37 ℃ of constant-temperature tables.
3) preparation of thrombus
By 20% urethane (6ml/kg for 350-400g male SD rat, i.p.), anesthesia, lies on the back fixing, separates right common carotid artery, bulldog clamp folder closes proximal part, the long polyethylene tube of 30mm is inserted in bulldog clamp top, emits about 3-4ml blood at every turn, approximately can put 2-3 time, immediately the blood of emitting is injected one by one and prepares the Glass tubing that thrombus is used with the 5ml syringe of silanization, stainless steel spiral is put at once.Standing 40min makes thrombosis, afterwards Glass tubing is carefully taken off from base, with fine needle by thrombus surrounding and Glass tubing inwall separately, removal of thromboses hangs on the rubber plug of reaction flask, in reaction flask, add 8ml distilled water, thrombus is suspended in water to standing 1 hour, remove the floating blood in thrombus surface.After 1 hour, with filter paper, suck the moisture on thrombus surface, 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), using physiological saline as blank, urokinase (100IU/ml) is as positive control, then thrombus hung in the solution of testing compound, and 37 ℃ of constant-temperature table 70rpm are hatched 2 hours.Hatch after end, with filter paper, draw surface water 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.Result is as shown in table 3.Result shows, 6a-f has outstanding external thrombolysis activity.
Table 3
Table 3 is the external thrombolysis activity of 10nM 6a-f a, wherein, a represents sample number, i.e. rat number used, and a) n=6, thrombus loss of weight represents with mean value ± SD mg; B) with physiological saline group ratio, p < 0.01.
The impact of experimental example 4 concentration on the external thrombus dissolving activity of 6d
According to the experimental technique of experimental example 3, choose the best 6d of thrombolytic effect and investigate 10nM, 1nM, the thrombolysis activity under tetra-concentration of 0.1nM and 0.01nM.Result is as shown in table 4.Result shows, the thrombolysis role in vitro display density dependency of 6d.
Table 4
Figure BSA00000374951600181
Table 4 is that the concentration of 6d is on the impact of external thrombolysis activity a, wherein, a represents sample number, i.e. rat number used, and a) n=6, thrombus loss of weight represents with mean value ± SD mg; B) with physiological saline and 1nM 6d group ratio, p < 0.01; C) with physiological saline and 0.1nM 6d group ratio, p < 0.01; D) with physiological saline and 0.01nM 6d group ratio, p < 0.01; E) with physiological saline group ratio, p > 0.05.
The particle diameter of the nanometer ball of 6a-f in experimental example 5 aqueous solution
1) particle diameter of 6a-f nanometer ball in the aqueous solution
While having observed 25 ℃ and 37 ℃ on laser light scattering particle size analyzer, 6a-f is 10 -4m, 10 -5m and 10 -6the particle diameter of the nanometer ball forming in the M aqueous solution.Result is as shown in table 5, and result shows, 6a-f can be assembled into nanometer ball in the aqueous solution, and in the time of 25 ℃, particle diameter is 57 to 777nm, and in the time of 37 ℃, particle diameter is 63 to 612nm.
Table 5
Figure BSA00000374951600182
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 drops in this solution on copper mesh, observes the form of nanometer ball after the dry solvent that volatilizees under JEM-1230 transmission electron microscope.Take 6d as example, the transmission electron microscope photo of 6d as shown in Figure 2.As can be seen from Figure 2, the nanometer ball of 6a-f formation rule.

Claims (5)

1. a compound of general formula 6a-f,
Wherein, n is 6,8,10,12,14 or 16.
2. a preparation method for general formula 6a-f compound described in claim 1, is characterized in that, comprises the steps:
(1) dicyclohexylcarbodiimide (DCC) exist under Boc-Pro in anhydrous THF and N-hydroxy-succinamide (HOSu) condensation be Boc-Pro-Osu, at NaHCO 3exist lower Boc-Pro-OSu to react with Ala and generate Boc-Pro-Ala;
(2) under DCC and HOBt existence, Boc-Pro-Ala is Boc-Pro-Ala-Lys (Z)-OBzl with Lys (Z)-OBzl condensation in anhydrous THF;
(3) under NaOH exists, in methyl alcohol, by Boc-Pro-Ala-Lys (Z)-OBzl saponification, be Boc-Pro-Ala-Lys (Z);
(4) under DCC exists, Boc-Asp (OBzl) is Boc-Asp (OBzl)-NHCH with the condensation of saturated fatty amine in anhydrous THF 2(CH 2) nCH 3; Described saturated fatty amine is CH 3(CH 2) n CH 2nH 2, wherein, n is 6,8,10,12,14 or 16;
(5) Boc-Asp (OBzl)-NHCH in hydrogenchloride-ethyl acetate solution 2(CH 2) nCH 3slough Boc and generate Asp (OBzl)-NH CH 2(CH 2) nCH 3;
(6) under DCC and HOBt exist Boc-Pro-Ala-Lys (Z) in anhydrous THF with Asp (OBzl)-NH-CH 2(CH 2) nCH 3condensation is Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NHCH 2(CH 2) nCH 3;
(7) NaOH exist under in methyl alcohol by Boc-Pro-Ala-Lys (Z)-Asp (OBzl)-NH-CH 2(CH 2) nCH 3saponification is Boc-Pro-Ala-Lys (Z)-Asp-NHCH 2(CH 2) nCH 3;
(8) Boc-Arg (NO under DCC and HOBt existence 2) in anhydrous THF, be Boc-Arg (NO with Gly-OBzl condensation 2)-Gly-OBzl;
(9) NaOH exist under in methyl alcohol by Boc-Arg (NO 2)-Gly-OBzl saponification is Boc-Arg (NO 2)-Gly;
(10) under DCC and HOBt existence, Boc-Asp (OBzl) is Boc-Asp (OBzl)-Ser (Bzl)-OBzl with Ser (Bzl)-OBzl condensation in anhydrous THF;
(11) in hydrogenchloride-ethyl acetate solution, Boc-Asp (OBzl)-Ser (Bzl)-OBzl sloughs Boc and generates Asp (OBzl)-Ser (Bzl)-OBzl;
(12) Boc-Arg (NO under DCC and HOBt existence 2)-Gly is Boc-Arg (NO with Asp (OBzl)-Ser (Bzl)-OBzl condensation in anhydrous THF 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl;
(13) Boc-Arg (NO in hydrogenchloride-ethyl acetate solution 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl sloughs Boc and generates Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl;
(14) Boc-Pro-Ala-Lys (Z)-Asp-NHCH under DCC and HOBt existence 2(CH 2) nCH 3in anhydrous THF with Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl condensation is Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) nCH 3;
(15) Boc-Pro-Ala-Lys (Z)-Asp[Arg (NO under trifluoromethanesulfonic acid and trifluoracetic acid existence 2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl]-NHCH 2(CH 2) nCH 3slough protecting group and generate general formula 6a-f compound.
3. a pharmaceutical composition, is characterized in that, the general formula 6a-f compound claimed in claim 1 that described pharmaceutical composition contains the upper effective dose for the treatment of, and contain one or more pharmaceutically acceptable excipient or additional dose.
4. a pharmaceutical preparation, is characterized in that, is the mixture of general formula 6a-f compound described in claim 1 and pharmaceutically acceptable excipient or additional dose is made to tablet, capsule, pulvis, granule, lozenge or oral liquid.
Described in claim 1 general formula 6a-f compound in the application of preparing in antithrombotic reagent.
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CN105884905A (en) 2012-09-05 2016-08-24 上海晟顺生物科技有限公司 Novel compound with thrombolytic, free radical scavenging and thrombus targeted functions, and preparation method and purposes thereof
CN105273052B (en) * 2014-06-19 2019-01-15 首都医科大学 Thrombus target discharges the synthesis and application of the poly- asparagus fern acyl-RGDS of antithrombotic agent of RGDS
CN108976280B (en) * 2017-05-30 2021-09-24 首都医科大学 Fatty amine modified LDV, its synthesis, activity and application

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