CN101906147B - Conjugate formed by two Arg-Gly-Asp-Ser chains and one fatty alcohol chain through Lys, synthesis thereof and application in medicine - Google Patents

Abstract

The invention discloses a conjugate formed by two Arg-Gly-Asp-Ser chains and one fatty alcohol chain through Lys, a synthesis thereof and an application in medicine. The general formula of the conjugate is Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH2-(CH2) nCH3, wherein n is equal to 6, 8, 10 or 12. The conjugate not only has the excellent oral anti-thrombosis activity, but also has excellent self-assembly performance, and can be used as a preparation material for preparing microemulsion, liposome and other drugs.

Description

Two Arg-Gly-Asp-Ser chains are through the conjugate of a Lys and an aliphatic alcohol chain, their the synthetic application that reaches in medical science

Technical field

The present invention relates to polypeptide-fatty alcohol couplet; Relate in particular to the conjugate that the carboxylic end coupling of saturated aliphatic chain alcohol through methoxyl group and nonapeptide gets; The compound method and this conjugate that the invention still further relates to this conjugate belong to biomedicine field as the application of pharmaceutical preparations having antithrombotic activity, drug carrier material.

Background technology

Gene therapies in 5 years to 10 years in future have the ideal carrier that can be sent to gene cell.See that from the position of biotechnological formulation the gene transfer system is exactly drug delivery system (DDS).The gene transfer system that has number of chemical synthetic small molecules to make up receives publicity, and wherein the gene transfer system of cation lipid formation is the focus of attention.Cation lipid at pharmaceutical field all over being used to make up liposomal delivery small molecules (comprising polypeptide) medicine.With the same in DDS; Cationic-liposome has obvious benefit in the gene transfer system, for example can through electric charge absorption form mixture with DNA and effectively avoid by lysosome degraded in the cell, can be with the efficient transfered cell of DNA, to the not restriction of size of DNA, easy and simple to handle etc.The essential structure of cation lipid is following:

The essential structure of cation lipid

Comprise hydrophobic arm and hydrophilic cationic head that senior aliphatic chain constitutes.

Cell adhesion plays a crucial role in the evolution of cell adhesion property disease (metastasis of cancer, thrombosis, chemistry cause inflammation and osteoporosis).Modulability gp for example RGD peptide and integrin receptor has very strong binding ability, can participate in the cell adhesion process.For example the RGD peptide combines to intervene thrombus, metastasis of cancer and osteoporotic evolution with the GP IIb/IIIa receptor-specific of thrombocyte, tumour cell and bone primary surface.This effect of RGD peptide has given the compound that contains the RGD sequence a kind of critical nature, and the compound that promptly contains the RGD sequence can be to thrombus, metastasis of cancer and osteoporotic disease sites enrichment.Under such prerequisite, RGD peptide and senior aliphatic chain coupling, the conjugate that just can obtain to expect.

Summary of the invention

One of the object of the invention provides the conjugate of following general formula: Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH ₂-(CH ₂) nCH ₃, n=6,8,10 or 12 wherein.

Two of the object of the invention provides a kind of method of synthetic above-mentioned conjugate.

A kind of method of synthetic above-mentioned conjugate may further comprise the steps:

Saturated aliphatic chain alcohol CH ₃(CH ₂) nCH ₂OH promptly gets through the carboxylic end coupling of methoxyl group and Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser) nonapeptide; Wherein n is selected from 6,8,10 or 12.

Preferably, a kind of method of synthetic above-mentioned conjugate may further comprise the steps:

(1) with the Methionin of tertbutyloxycarbonyl protection respectively with CH ₃(CH ₂) ₆CH ₂OH, CH ₃(CH ₂) ₈CH ₂OH, CH ₃(CH ₂) ₁₀CH ₂OH, CH ₃(CH ₂) ₁₂CH ₂The OH coupling;

(2) slough the tertbutyloxycarbonyl of step (1) gained conjugate;

(3) Serine and step (2) products therefrom with the tertbutyloxycarbonyl protection carries out coupling;

(4) slough the tertbutyloxycarbonyl of step (3) gained conjugate;

The aspartic acid and step (4) the products therefrom coupling of (5) holding tertbutyloxycarbonyl protection side chain benzyloxy to protect N;

(6) slough the tertbutyloxycarbonyl of step (5) gained conjugate;

(7) N is held the l-arginine of tertbutyloxycarbonyl protection side chain nitro protection and the glycocoll coupling of benzyloxy protection;

(8) with (6) and (7) products therefrom coupling; Remove the protection base, promptly get.

The present invention has combined following understanding or foundation to accomplish technique scheme.The ideal carrier (gene transfer system) that is sent to gene in cell is one of mission critical of gene therapy; See that from the position of biotechnological formulation gene transfer system and drug delivery system (DDS) have identity property; The gene transfer system that cation lipid constitutes though the gene transfer system that has number of chemical synthetic small molecules to make up all receives publicity should especially pay close attention; Cationic-liposome can form mixture with DNA through electric charge absorption and also effectively avoid by lysosome degraded in the cell; Cationic-liposome can not limit the efficient transfered cell of DNA, cationic-liposome to the size of DNA; Thrombocyte, inflammation tissue, cancer cells and ground substance of bone show and are rich in the cell adhesion acceptor that can discern the RGD sequence that the compound that contains the RGD sequence can be to thrombus, inflammation, metastasis of cancer and osteoporotic disease sites enrichment.

The inventor is based on above-mentioned cognition, saturated aliphatic chain alcohol CH ₃(CH ₂) nCH ₂OH (n is selected from 6,8,10 or 12) makes the molecule that makes obtain four kinds of performances through the carboxylic end coupling of methoxyl group and Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser) nonapeptide, promptly depends on saturated aliphatic chain alcohol CH ₃(CH ₂) nCH ₂The cationic property of the hydrophobicity of OH, the guanidine radicals that depends on the RGD peptide and the protonated formation of alpha-amino group and hydrophilic depends on the target property that the RGD peptide obtains GPIIb/IIIa receptor-specific affinity interaction.Under such prerequisite, pass through the Lys coupling to Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser) nonapeptide and saturated aliphatic chain alcohol, just can obtain the conjugate of an Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser) and an aliphatic alcohol chain.

The evaluation that forms on the model at rat suppository shows that conjugate of the present invention has the excellent oral antithrombotic acitivity, can be used as pharmaceutical preparations having antithrombotic activity and uses; Detected conjugate of the present invention at water and be self-assembled into the performance into nano particle, test-results shows that conjugate of the present invention has outstanding self-assembly performance, can be used as the formulation materials of preparation micro emulsion, liposome etc.

Another purpose of the present invention provides a kind of pharmaceutical composition that contains conjugate of the present invention, and this pharmaceutical composition is gone up effective dose by treatment conjugate of the present invention is with pharmaceutically acceptable excipient or assist and add agent and form; That is: with the conjugate of the present invention of significant quantity with after pharmaceutically acceptable carrier or thinner cooperate, the formulation method conventional by this area is prepared into any one appropriate drug compsn with it.Usually said composition is suitable for oral administration and drug administration by injection, also is fit to other medication.Said composition can be liquid preparation forms such as tablet, capsule, pulvis, granule, lozenge, suppository, or oral liquid.According to different medications, pharmaceutical composition of the present invention can contain 0.1%-99% weight, the conjugate of the present invention of preferred 10-60% weight.

Description of drawings

The synthetic route chart of Fig. 1 conjugate of the present invention; I) DCC, HOBt, NMM; Ii) 4N HCl-EtOAc; Iii) H ₂, Pd/C; N=6,8,10 or 12 wherein.

The nanometer ball that Fig. 2 is assembled at water by the representative compound of the present invention that transmission electron microscope is described.

The nanoparticle state of representative compound of the present invention after water assembling freeze-drying that Fig. 3 is described by ESEM.

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.

Boc-Arg (NO ₂The preparation of)-Gly-OMe

With Boc-Arg (NO ₂) 1.60g (5mmol) is dissolved in the anhydrous THF of 20ml; Add N-hydroxy benzo triazole (HOBt) 0.68g (5mmol) under the condition of ice bath, and make dissolving fully, add dicyclohexyl carbonyl diimine (DCC) 1.07g (6mmol) after 10 minutes; Stir 30min, obtain reaction solution (i).Under the condition of ice bath Gly-OMe 0.53 (6mmol) is suspended among the anhydrous THF of 20ml, adds 1mlN-methylmorpholine (NMM), transfer pH 8-9.Stir 30min, obtain reaction solution (ii), add in the reaction solution (i), ice bath stirs 1h, stirring at room 12h again, TLC (chloroform: methyl alcohol=10: 1) show Boc-Arg (NO ₂) disappear.Filtering NSC 30023 (DCU), pressure reducing and steaming THF.Residue is used the 50ml acetic acid ethyl dissolution, and gained solution is used 5%NaHCO successively ₃The aqueous solution, the saturated NaCl aqueous solution, 5%KHSO ₄The aqueous solution is washed, the saturated NaCl aqueous solution, saturated NaHCO ₃The aqueous solution, the saturated NaCl aqueous solution are given a baby a bath on the third day after its birth inferior, and ethyl acetate layer is used anhydrous Na ₂SO ₄Drying is crossed the filtering siccative, and filtrate decompression is concentrated into dried, obtains title compound 1.78g (yield 91.3%).Rf=0.39 (chloroform: .ESI-MS (m/z) methyl alcohol=10: 1): 391 [M+H] ⁺, 490 [M+Na] ⁺

Boc-Arg (NO ₂The preparation of)-Gly

With Boc-Arg (NO ₂)-Gly-OMe 1.95g (5mmol) is dissolved in 10ml methyl alcohol.Under the ice bath gained solution is transferred pH12 and stirred 2h with the 2N NaOH aqueous solution, TLC (chloroform: methyl alcohol=15: 1) show Boc-Arg (NO ₂)-Gly-OMe disappears.Reaction mixture is used saturated KHSO ₄The aqueous solution is transferred pH7, and concentrating under reduced pressure is removed methyl alcohol.Residual solution is used saturated KHSO ₄The aqueous solution is transferred pH2, ethyl acetate extraction (30ml * 3).The ETHYLE ACETATE that merges is washed till neutrality, anhydrous Na with the saturated NaCl aqueous solution mutually ₂SO ₄Dry.Filter, filtrate decompression is concentrated into dried, gets title compound colorless solid 1.80g (yield 95.8%). ${\left[\mathrm{\α}\right]}_D^{20}=-2.16(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ Rf=0.12 (chloroform: .ESI-MS (m/z) methyl alcohol=2: 1): 375 [M-H] ⁺.

Embodiment 1Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH ₂(CH ₂) ₆CH ₃Preparation (9a)

1)Boc-Lys(Boc)-OCH ₂-(CH ₂) ₆CH ₃(1a)

1.82g (5.25mmol) Boc-Lys (Boc) is dissolved among the anhydrous THF of 20ml; Ice bath adds 0.75g (5.5mmol) N-hydroxy benzo triazole (HOBt) down; Add 1.23g (6mmol) dicyclohexyl carbonyl diimine (DCC) after 10 minutes, obtain reaction solution (i), for use.The following 0.65g of ice bath (5mmol) CH ₃(CH ₂) ₆CH ₂OH is dissolved among the anhydrous THF of 20ml, obtains reaction solution (ii), and is for use.The following reaction solution of ice bath (ii) adds in the reaction solution (i), and first ice bath stirs down 1h, stirring at room 12h again, and TLC shows that Boc-Lys (Boc) disappears.Filtering NSC 30023 (DCU), filtrate decompression concentrate yellow oil, the 50ml acetic acid ethyl dissolution leaches the DCU that separates out once more, filtrates and uses 5%NaHCO successively ₃The aqueous solution, the saturated NaCl aqueous solution, 5%KHSO ₄The aqueous solution, the saturated NaCl aqueous solution, 5%NaHCO ₃The aqueous solution, the saturated NaCl aqueous solution are respectively washed 3 times.Ethyl acetate layer is used anhydrous Na ₂SO ₄Drying, filtration, filtrate decompression are concentrated into to do and promptly get title compound, are colourless oil liquid.ESI-MS(m/z)：459[M+H] ⁺

2)Lys-OCH ₂-(CH ₂) ₆CH ₃(2a)

With 0.46g (1mmol) Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Use the 2ml acetic acid ethyl dissolution; Ice bath stirs and adds the reaction of 10ml4N HCl/ ethyl acetate solution down, behind the 2h, and TLC (sherwood oil: ETHYLE ACETATE=2: 1) show that raw material point disappears; 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 hydrogen chloride gas.Add a small amount of sherwood oil at last residue is ground to form 0.32g (95.8%) title compound, be colorless solid, directly be used for next step reaction.ESI-MS(m/z)：259[M+H] ⁺

3)Boc-Ser(Bzl)-Lys[Boc-Ser(Bzl)]-OCH ₂-(CH ₂) ₆CH ₃(3a)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method with 1.47g (5mmol) Boc-Ser (Bzl) and 0.79g (2.4mmol) Lys-OCH ₂-(CH ₂) ₆CH ₃Make title compound 1.65g (84.7%), be colorless solid. ${\left[\mathrm{\α}\right]}_D^{20}=11.57(C=0.7,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.8-79℃.ESI-MS(m/z)：813[M+H] ⁺.

4)Ser(Bzl)-Lys[Ser(Bzl)]-OCH ₂-(CH ₂) ₆CH ₃(4a)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with 0.81g (1mmol) Ser (Bzl)-Lys [Ser (Bzl)]-OCH ₂-(CH ₂) ₆CH ₃Make title compound 0.65g (94.9%), be faint yellow solid, directly be used for next step reaction.ESI-MS(m/z)：613[M+H] ⁺.

5)Boc-Asp(OBzl)-Ser(Bzl)-Lys[Boc-Asp(OBzl)-Ser(Bzl)]-OCH ₂(CH ₂) ₆CH ₃(5a)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method with 1.61g (5mmol) Boc-Asp (OBzl) and 1.64g (2.4mmol) Ser (Bzl)-Lys [Ser (Bzl)]-OCH ₂-(CH ₂) ₆CH ₃Make title compound 2.86g (97.5%), be colorless solid. ${\left[\mathrm{\α}\right]}_D^{20}=2.80(C=0.95,{\mathrm{CH}}_3\mathrm{OH}),$ M.p.7-58℃.ESI-MS(m/z)：1223[M+H] ⁺，

6)Asp(OBzl)-Ser(Bzl)-Lys[Asp(OBzl)-Ser(Bzl)]-OCH ₂(CH ₂) ₆CH ₃(6a)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with 1.22g (1mmol) Boc-Asp (OBzl)-Ser (Bzl)-Lys [Boc-Asp (OBzl)-Ser (Bzl)]-OCH ₂-(CH ₂) ₆CH ₃Make 1.05g (95.9%) title compound, be colorless solid, directly be used for next step reaction.ESI-MS(m/z)：1023[M+H] ⁺

7)Boc-Arg(NO ₂)-Gly-Asp(OBzl)-Ser(Bzl)-Lys[Boc-Arg(NO ₂)-Gly-Asp(OBzl)-Ser(Bzl)]-OCH ₂(CH ₂) ₆CH ₃(7a)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method with 1.88g (5mmol) Boc-Arg (NO ₂)-Gly and Asp (OBzl)-Ser (Bzl)-Lys [Asp (OBzl)-Ser (Bzl)]-OCH ₂-(CH ₂) ₆CH ₃2.63g (2.4mmol) get title compound 2.88g (68.9%), be colorless solid. ${\left[\mathrm{\α}\right]}_D^{20}=-10.12(C=0.8,{\mathrm{CH}}_3\mathrm{OH}),$ M.p.109-112℃.ESI-MS(m/z)：1740[M+H] ⁺.

8)Boc-Arg-Gly-Asp-Ser-Lys(Boc-Arg-Gly-Asp-Ser)-OCH ₂-(CH ₂) ₆CH ₃(8a)

Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-Lys [Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)]-OCH ₂-(CH ₂) ₆CH ₃0.17g (0.1mmol) place the 50ml eggplant-shape bottle, use dissolve with methanol, add 50mgPd/C (30%), logical H ₂, stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression is concentrated into dried, and residue grinds with ether repeatedly, makes 0.096g (88.0%) title compound, is the colorless solid powder.ESI-MS(m/z)：1288[M-H] ⁺

9)Arg-Gly-Asp-Ser-Lys(Arg-Gly-Asp-Ser)-OCH ₂(CH ₂) ₆CH ₃(9a)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with 1.29g (1mmol) Boc-Arg-Gly-Asp-Ser-Lys (Boc-Arg-Gly-Asp-Ser)-OCH ₂-(CH ₂) ₆CH ₃Make Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH ₂(CH ₂) ₆CH ₃1.01g (92.7%), be the colorless solid powder. ${\left[\mathrm{\α}\right]}_D^{20}=-23.29(C=0.9,{\mathrm{CH}}_3\mathrm{OH}),$ M.p.178-179℃.ESI-MS(m/z)：1089[M+H] ⁺

Embodiment 2Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH ₂(CH ₂) ₈CH ₃Preparation (9b)

1) Boc-Lys (Boc)-OCH ₂-(CH ₂) ₈CH ₃Preparation (1b)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method obtain title compound 2.40g (yield 98.7%) with Boc-Lys (Boc) 1.73g (5mmol) and positive ten pure 0.95g (6mmol). ${\left[\mathrm{\α}\right]}_D^{20}=12.33(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ Rf=0.47 (sherwood oil: .ESI-MS (m/z) acetone=7: 1): 509 [M+Na] ⁺.

2) Lys-OCH ₂-(CH ₂) ₈CH ₃Preparation (2b)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with Boc-Lys (Boc)-OCH ₂-(CH ₂) ₈CH ₃0.49g (1mmol) getting colorless solid is title compound 0.31g (yield 84.9%).ESI-MS(m/z)：287[M+H] ⁺，573[2M+H] ⁺.

3) Boc-Ser (Bzl)-Lys [Boc-Ser (Bzl)]-OCH ₂(CH ₂) ₈CH ₃Preparation (3b)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method with Boc-Ser (Bzl) 0.62g (2.1mmol) and Lys-OCH ₂-(CH ₂) ₈CH ₃0.36g (1mmol) obtain title compound 0.79g (yield 94.2%). ${\left[\mathrm{\α}\right]}_D^{20}=-4.83(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.:77-79 ℃ of .Rf=0.32 (sherwood oil: .ESI-MS (m/z) acetone=4: 1): 841 [M+H] ⁺.

4) Ser (Bzl)-Lys [Ser (Bzl)]-OCH ₂(CH ₂) ₈CH ₃Preparation (4b)

According to preparation L-ysOCH ₂-(CH ₂) ₆CH ₃Method with Boc-Ser (Bzl)-Lys [Boc-Ser (Bzl)]-OCH ₂-(CH ₂) ₈CH ₃0.84g (1mmol) get colorless solid, promptly get title compound 0.70g (yield 98.2%).ESI-MS(m/z)：641[M+H] ⁺.

5) Boc-Asp (OBzl)-Ser (Bzl)-Lys [Boc-Asp (OBzl)-Ser (Bzl)]-OCH ₂(CH ₂) ₈CH ₃Preparation (5b)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method with Ser (Bzl)-Lys [Ser (Bzl)]-OCH ₂-(CH ₂) ₈CH ₃0.71g (1mmol) obtain title compound 1.07g (yield 85.8%). ${\left[\mathrm{\α}\right]}_D^{20}=-16.00(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.:52-54 ℃ of .Rf=0.30 (sherwood oil: .ESI-MS (m/z) acetone=1: 1): 1273 [M+Na] ⁺.

6) Asp (OBzl)-Ser (Bzl)-Lys [Asp (OBzl)-Ser (Bzl)]-OCH ₂(CH ₂) ₈CH ₃Preparation (6b)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with Boc-Asp (OBzl)-Ser (B zl)-Lys [Boc-Asp (OBzl)-Ser (Bzl)]-OCH ₂-(CH ₂) ₈CH ₃1.25g (1mmol) get colorless solid, promptly get title compound 0.99g (yield 94.10%).ESI-MS(m/z)：1051[M+H] ⁺.

7) Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-Lys [Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)]-OCH ₂(CH ₂) ₈CH ₃Preparation (7b)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method with Asp (OBzl)-Ser (Bzl)-Lys [Asp (OBzl)-Ser (Bzl)]-OCH ₂-(CH ₂) ₈CH ₃1.05g (1mmol) obtain title compound 1.29g (yield 73.5%). ${\left[\mathrm{\α}\right]}_D^{20}=-15.63(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ Mp 97-99 ℃ .Rf=0.15 (chloroform: .ESI-MS (m/z) methyl alcohol=20: 1): 1790 [M+Na] ⁺.

8) Boc-Arg-Gly-Asp-Ser-Lys (Boc-Arg-Gly-Asp-Ser)-OCH ₂-(CH ₂) ₈CH ₃Preparation (8b)

Adopt and preparation Boc-Arg-Gly-Asp-Ser-Lys (Boc-Arg-Gly-Asp-Ser)-OCH ₂-(CH ₂) ₆CH ₃Identical method is with Boc-Arg-Gly-Asp-Ser-Lys (Boc-Arg-Gly-Asp-Ser)-OCH ₂-(CH ₂) ₈CH ₃0.88g (0.5mmol) get title compound 0.70g (productive rate 80.3%).ESI-MS(m/z)：1315[M-H] ^-.

9) Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH ₂(CH ₂) ₈CH ₃Preparation (9b)

According to preparation LysOCH ₂(CH ₂) ₆CH ₃Method with 0.66mg (0.5mmol) (Boc-Arg-Gly-Asp-Ser-) ₂LysOCH ₂(CH ₂) ₈CH ₃Get colorless solid, the desalination of Sephadex G10 column chromatography purification promptly gets title compound 0.50g (yield 90.1%). ${\left[\mathrm{\α}\right]}_D^{20}=-21.93(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ Mp 194-195 ℃ .Rf=0.16 (propyl carbinol: Glacial acetic acid min. 99.5: .ESI-MS (m/z) water=4: 1: 1): 1116 [M-H] ^-.

Embodiment 3Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH ₂(CH ₂) ₁₀CH ₃Preparation 1 (9c)) Boc-Lys (Boc)-OCH ₂(CH ₂) ₁₀CH ₃Preparation (1c)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method obtain title compound 2.55g (yield 99.0%) with lauryl alcohol 1.116g (6mmol). ${\left[\mathrm{\α}\right]}_D^{20}=-9.20(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ Rf=0.49 (sherwood oil: .ESI-MS (m/z) acetone=7: 1): 515 [M+H] ⁺, 537 [M+Na] ⁺.

2) Lys-OCH ₂-(CH ₂) ₁₀CH ₃Preparation (2c)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with Boc-Lys (Boc)-OCH ₂-(CH ₂) ₁₀CH ₃0.51g (1mmol) get colorless solid, promptly get title compound 0.36g (yield 92.7%).ESI-MS(m/z)：315[M+H] ⁺.

3) Boc-Ser (Bzl)-Lys [Boc-Ser (Bzl)]-OCH ₂(CH ₂) ₁₀CH ₃Preparation (3c)

According to preparation Boc-Lys (Boc)-OCH ₂(CH ₂) ₆CH ₃Method with Lys-OCH ₂-(CH ₂) ₁₀CH ₃0.39g (1mmol) obtain title compound 0.84g (yield 96.2%). ${\left[\mathrm{\α}\right]}_D^{20}=-4.00(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.:87-89 ℃ of .Rf=0.38 (sherwood oil: .ESI-MS (m/z) acetone=3: 1): 870 [M+H] ⁺, 892 [M+Na] ⁺.

4) Ser (Bzl)-Lys [Ser (Bzl)]-OCH ₂-(CH ₂) ₁₀CH ₃Preparation (4c)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with Boc-Ser (Bzl)-Lys [Boc-Ser (Bzl)]-OCH ₂-(CH ₂) ₁₀CH ₃0.87g (1mmol) get colorless solid, promptly get title compound 0.72g (yield 97.8%).ESI-MS(m/z)：669[M+H] ⁺.

5) Boc-Asp (OBzl)-Ser (Bzl)-Lys [Boc-Asp (OBzl)-Ser (Bzl)]-OCH ₂(CH ₂) ₁₀CH ₃Preparation (5c)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method with Ser (Bzl)-Lys [Ser (Bzl)]-OCH ₂-(CH ₂) ₁₀CH ₃0.74g (1mmol) obtain title compound 1.20g (yield 94.1%). ${\left[\mathrm{\α}\right]}_D^{20}=-18.63(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.:81-83 ℃ of .Rf=0.31 (sherwood oil: .ESI-MS (m/z) acetone=2: 1): 1302 [M+Na] ⁺.

6) Asp (OBzl)-Ser (Bzl)-Lys [Asp (OBzl)-Ser (Bzl)]-OCH ₂-(CH ₂) ₁₀CH ₃Preparation (6c)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with Boc-Asp (OBzl)-Ser (Bzl)-Lys [Boc-Asp (OBzl)-Ser (Bzl)]-OCH ₂-(CH ₂) ₁₀CH ₃1.28mg (1mmol) get colorless solid, promptly get title compound 1.09mg (yield 94.4%).ESI-MS(m/z)：1080[M+H] ⁺.

7) Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-Lys [Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)]-OCH ₂-(CH ₂) ₁₀CH ₃Preparation (7c)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method with Asp (OBzl)-Ser (Bzl)-Lys [Asp (OBzl)-Ser (Bzl)]-OCH ₂(CH ₂) ₁₀CH ₃1.08g (1mmol) obtain title compound 1.27g (yield 70.5%). ${\left[\mathrm{\α}\right]}_D^{20}=-17.07(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.:94-96 ℃ of .Rf=0.44 (chloroform: .ESI-MS (m/z) methyl alcohol=10: 1): 1818 [M+Na] ⁺.

8) Boc-Arg-Gly-Asp-Ser-Lys (Boc-Arg-Gly-Asp-Ser)-OCH ₂-(CH ₂) ₁₀CH ₃Preparation (8c)

Adopt and preparation (Boc-Arg-Gly-Asp-Ser-) ₂LysOCH ₂(CH ₂) ₆CH ₃Identical method is with Boc-Arg-Gly-Asp-Ser-Lys (Boc-Arg-Gly-Asp-Ser)-OCH ₂-(CH ₂) ₆CH ₃0.90g (0.5mmol) get title compound 0.58g (productive rate 85.8%).ESI-MS(m/z)：1344[M-H] ^-.

9) Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH ₂(CH ₂) ₁₀CH ₃Preparation (9c)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with Boc-Arg-Gly-Asp-Ser-Lys (Boc-Arg-Gly-Asp-Ser)-OCH ₂(CH ₂) ₁₀CH ₃0.67g (0.5mmol) get colorless solid 0.52g (yield 91.2%). ${\left[\mathrm{\α}\right]}_D^{20}=-18.03(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.186-188 ℃ of .Rf=0.18 (propyl carbinol: Glacial acetic acid min. 99.5: .ESI-MS (m/z) water=4: 1: 1): 1146 [M+H] ⁺.

Embodiment 4Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH ₂(CH ₂) ₁₂CH ₃Preparation (9d)

1) Boc-Lys (Boc)-OCH ₂-(CH ₂) ₁₂CH ₃Preparation (1d)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method obtain title compound 2.63g (yield 97.2%) with tetradecyl alcohol 1.28g (6mmol). ${\left[\mathrm{\α}\right]}_D^{20}=-16.73(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.:29-31 ℃ of .Rf=0.44 (sherwood oil: .ESI-MS (m/z) acetone=8: 1): 543 [M+H] ⁺.

2) Lys-OCH ₂-(CH ₂) ₁₂CH ₃Preparation (2d)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with Boc-Lys (Boc)-OCH ₂(CH ₂) ₁₂CH ₃0.54g (1mmol), getting title compound 0.32g (yield 94.4%) is colorless solid.ESI-MS(m/z)：343[M+H] ⁺.

3) Boc-Ser (Bzl)-Lys [Boc-Ser (Bzl)]-OCH ₂-(CH ₂) ₁₂CH ₃Preparation (3d)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method with Lys-OCH ₂-(CH ₂) ₁₂CH ₃0.36g (1mmol) obtain title compound 0.88g (yield 97.8%). ${\left[\mathrm{\α}\right]}_D^{20}=1.90(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.:76-78 ℃ of .Rf=0.45 (sherwood oil: .ESI-MS (m/z) acetone=3: 1): 920 [M+Na] ⁺.

4) Ser (Bzl)-Lys [Ser (Bzl)]-OCH ₂-(CH ₂) ₁₂CH ₃Preparation (4d)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with Boc-Ser (Bzl)-Lys [Boc-Ser (Bzl)]-OCH ₂(CH ₂) ₁₂CH ₃0.90g (1mmol) get colorless solid, promptly get title compound 0.75g (yield 97.9%).ESI-MS(m/z)：697[M+H] ⁺.

5) Boc-Asp (OBzl)-Ser (Bzl)-Lys [Boc-Asp (OBzl)-Ser (Bzl)]-OCH ₂(CH ₂) ₁₂CH ₃Preparation (5d)

According to preparation Boc-Lys (Boc)-OCH ₂(CH ₂) ₆CH ₃Method with Boc-Ser (Bzl)-Lys [Boc-Ser (Bzl)]-OCH ₂(CH2) ₁₂CH ₃0.77g (1mmol) obtain title compound 1.28g (yield 97.8%). ${\left[\mathrm{\α}\right]}_D^{20}=-14.03(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.:84-85 ℃ of .Rf=0.44 (sherwood oil: .ESI-MS (m/z) acetone=2: 1): 1330 [M+Na] ⁺.

6) Asp (OBzl)-Ser (Bzl)-Lys [Asp (OBzl)-Ser (Bzl)]-OCH ₂-(CH ₂) ₁₂CH ₃Preparation (6d)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with Boc-Asp (OBzl)-Ser (Bzl)-Lys [Boc-Asp (OBzl)-Ser (Bzl)]-OCH ₂(CH ₂) ₁₂CH ₃1.31g (1mmol) get colorless solid, i.e. title compound 1.13g (yield 95.8%).ESI-MS(m/z)：1108[M+H] ⁺，1146[M+Na] ⁺.

7) Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-Lys [Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)]-OCH ₂(CH ₂) ₁₂CH ₃Preparation (7d)

According to preparation Boc-Lys (Boc)-OCH ₂-(CH ₂) ₆CH ₃Method with Asp (OBzl)-Ser (Bzl)-Lys [Asp (OBzl)-Ser (Bzl)]-OCH ₂(CH ₂) ₁₂CH ₃1.18g (1mmol) obtain title compound 1.36g (yield 74.3%). ${\left[\mathrm{\α}\right]}_D^{20}=-18.07(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.:98-99 ℃ of .Rf=0.15 (chloroform: .ESI-MS (m/z) methyl alcohol=20: 1): 1846 [M+Na] ⁺, 1862 [M+K] ⁺.

8) Boc-Arg-Gly-Asp-Ser-Lys [Boc-Arg-Gly-Asp-Ser]-OCH ₂-(CH ₂) ₁₂CH ₃Preparation (8d)

Adopt [Boc-Arg-Gly-Asp-Ser-OCH with preparation Boc-Arg-Gly-Asp-Se-Lys ₂-(CH ₂) ₁₂CH ₃Identical method is with Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-Lys [Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)]-OCH ₂(CH ₂) ₁₂CH ₃0.92g (0.5mmol) get title compound 0.58g (productive rate 84.3%).ESI-MS(m/z)：1372[M-H] ⁺.

9) Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH ₂(CH ₂) ₁₂CH ₃Preparation (9d)

According to preparation Lys-OCH ₂-(CH ₂) ₆CH ₃Method with Boc-Arg-Gly-Asp-Ser-Lys [Boc-Arg-Gly-Asp-Ser]-OCH ₂-(CH ₂) ₁₂CH ₃0.69mg (0.5mmol) get colorless solid 0.54g (yield 92.7%). ${\left[\mathrm{\α}\right]}_D^{20}=-14.13(C=1,{\mathrm{CH}}_3\mathrm{OH}).$ M.p.:202-204 ℃ of .Rf=0.20 (propyl carbinol: Glacial acetic acid min. 99.5: .ESI-MS (m/z) water=4: 1: 1): 1173 [M+H] ⁺.

The antithrombotic acitivity test of Test Example 1 The compounds of this invention oral administration

1) dosage

Frosst) and The compounds of this invention are mixed with physiological salt soln respectively before administration.Be divided into three groups; Empty no control group, Frosst) group and test group; Wherein, (dosage is 12mg/kg to the physiological salt soln of empty no control group orally give saline water (dosage is 3ml/kg), Frosst) group orally give Frosst), 66umol/kg), the physiological salt soln (dosage is 1nmol/kg) of test group orally give The compounds of this invention.

2) rat operation and apparatus

(male, 190～210g) anaesthetize by 1.2g/kg dosage abdominal injection urethane solution the Wistar rat.The anesthetized rat dorsal position is fixed, and separates RCCA, the left side vein end, and in proximal part folder bulldog clamp, proximal part and distal end penetrate surgical thread respectively separately, with the surgical thread ligation of distal end, prepare intubate.

3) intubate

Intubate is the polyethylene rubber tube that silylanization is crossed, and divides three sections, and the stage casing is a polyethylene rubber tube, long 60.0mm, internal diameter 3.5mm; Two ends are identical polyethylene tube, and pipe range 100.0mm, the end that internal diameter 1.0mm, external diameter 2.0mm should manage pull into point pipe (being used to insert rat carotid artery or vein), and external diameter is 1.0mm.Be respectively charged into the long black surgical thread of 6cm in the EP pipe with the number of finishing, weigh; Take out silk thread then, put into the thicker intubate in stage casing of ready intubate according to numbering.

Behind the gastric infusion 30 minutes, rat separates right carotid and left jugular vein with 20% urethane solution (0.7ml/100g) anesthesia back, and folder closes right carotid artery; The ligation of artery distal end is full of the physiological salt soln (84mg/200ml saline water) of heparin sodium with intubate, and an end inserts the left side vein; After the other end adds quantitative heparin sodium anti-freezing with syringe; Insert the right side artery, open bulldog clamp, make blood begin circulation.Take out after 15 minutes with the silk thread of thrombus and put back to corresponding EP pipe and record weight, (NS 3ml/kg) makes blank, and (60mg/15ml 3ml/kg) makes positive control with Frosst) with saline water.Can produce thrombus because of blood circulation on the silk thread in this process in the extra heavy pipe of intubate central authorities.

4) thrombus is weighed

Timing is cut off venous incubation after beginning 15 minutes, stops circulation, carefully takes out silk thread with the ophthalmology tweezer, on filter paper, dips in drop of blood gently, puts into the EP pipe of weighing in advance, accurately weighs and record.Calculate the weight in wet base of thrombus.Each medicine repeats 10 administrations.The wet weight of thrombus of each group of statistics (X ± SD), and do the t check.

5) result

The result lists table 1 in.The result shows that compound of the present invention has good antithrombotic acitivity under 1nmol/kg dosage.It is thus clear that The compounds of this invention all is outstanding pharmaceutical preparations having antithrombotic activity.

The antithrombotic acitivity evaluation of table 1 The compounds of this invention oral administration administration

Compound	Wet weight of thrombus ( ±SD?mg)
		NS	32.74±2.98
Frosst)	14.43±2.90 a
		9a	17.07±2.83 a
9b	15.72±3.85 a
		9c	19.95±1.16 a
9d	12.24±3.62 a

N=10; NS=saline water; A compares with the saline water group, P＜0.01;

Test Example 2 The compounds of this invention are in the test of aqueous phase assembling becoming nanometer ball

1) 4 kinds of positively charged ion pharmacomes of the present invention are according to 10 ^-3The concentration of mol/L is configured as the aqueous solution, goes up METHOD FOR CONTINUOUS DETERMINATION 8 days at laser light scattering nano particle size appearance (ZetaPlus JEM-1230), observes size and stability thereof, and the result sees table 2 and table 3.The data that obtain show that these 4 kinds of positively charged ion pharmacomes can be self-assembled at aqueous phase and be the stabilized nano ball, thereby are the outstanding micro emulsion and the preparation material of liposome medicament.

Table 24 kinds of positively charged ion pharmacomes of the present invention are self-assembled into the particle diameter into nanometer ball in water

Compound	Particle diameter (nm)
		9a	198

9b	153
		9c	143
9d	152

Table 34 kinds of positively charged ion pharmacomes of the present invention are self-assembled into the particle diameter stability into nanometer ball in water

2) according to the mensuration requirement of transmission electron microscope, the aqueous solution of 4 kinds of conjugates of configuration places the dry back of copper mesh Back stroke to measure the figure of compound of the present invention at the nanometer ball of water assembling.The result shows, under transmission electron microscope, can see uniform granular attitude by the nanometer ball of these 4 kinds of conjugate assemblings.Given here is representative compound

Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH ₂-(CH ₂) ₁₂CH ₃(Fig. 2)

3) according to the mensuration requirement of ESEM, dispose the aqueous solution of 4 kinds of conjugates, measure the microtexture and the particle diameter of compound of the present invention after the lyophilize.The result shows, under ESEM, can be seen spherical and bar-shaped and deposited by the nanoparticle of these 4 kinds of conjugates assemblings.Given here is representative compound Arg-Gly-Asp-Ser-Lys (Arg-Gly-Asp-Ser)-OCH ₂-(CH ₂) ₁₀CH ₃(Fig. 3)

Claims (6)

1. the conjugate of formula:

be n=6,8,10 or 12 wherein.

2. the method for the described conjugate of synthetic claim 1 may further comprise the steps:

Saturated aliphatic chain alcohol CH ₃(CH ₂) nCH ₂CH among the OH ₃(CH ₂) CH of n group through thereafter ₂The carboxyl terminal of OH and Arg-Gly-Asp-Ser-Lys-Ser-Asp-Gly-Arg nonapeptide Methionin carries out coupling through forming methoxyl group, promptly gets; Wherein n is selected from 6,8,10 or 12.

3. the method for the described conjugate of synthetic claim 1 may further comprise the steps:

(1) with the Methionin of tertbutyloxycarbonyl protection respectively with CH ₃(CH ₂) ₆CH ₂OH, CH ₃(CH ₂) ₈CH ₂OH, CH ₃(CH ₂) ₁₀CH ₂OH, CH ₃(CH ₂) ₁₂CH ₂The OH coupling;

(2) slough the tertbutyloxycarbonyl of step (1) gained conjugate;

(3) Serine and step (2) products therefrom with the tertbutyloxycarbonyl protection carries out coupling;

(4) slough the tertbutyloxycarbonyl of step (3) gained conjugate;

The aspartic acid and step (4) the products therefrom coupling of (5) holding tertbutyloxycarbonyl protection, side chain benzyloxy to protect N;

(6) slough the tertbutyloxycarbonyl of step (5) gained conjugate;

(7) N is held the l-arginine of tertbutyloxycarbonyl protection, the protection of side chain nitro and the glycocoll coupling of benzyloxy protection;

(8) with (6) and (7) products therefrom coupling, remove the protection base, promptly get.

4. antithrombotic pharmaceutical composition is characterized in that: the described conjugate of claim 1 and pharmaceutically acceptable carrier or the auxiliary material of being gone up significant quantity by treatment or prevention are formed.

5. the described conjugate of claim 1 is in the purposes of preparation in the antithrombotic reagent.

6. the purposes of the described conjugate of claim 1 in the formulation materials of preparation micro emulsion, liposome medicament.

Images