CN101899090A - Saturated fatty chain alcohol His-Gly-AA tripeptide ester, synthetic method and application thereof - Google Patents

Saturated fatty chain alcohol His-Gly-AA tripeptide ester, synthetic method and application thereof Download PDF

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CN101899090A
CN101899090A CN2009100853253A CN200910085325A CN101899090A CN 101899090 A CN101899090 A CN 101899090A CN 2009100853253 A CN2009100853253 A CN 2009100853253A CN 200910085325 A CN200910085325 A CN 200910085325A CN 101899090 A CN101899090 A CN 101899090A
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CN101899090B (en
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赵明
彭师奇
赵淑锐
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Capital Medical University
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Abstract

The invention relates to 12 saturated fatty chain alcohol His-Gly-AA tripeptide ester conjugates with immunosuppressive activity in a general formula His-Gly-AA-O-CH2-(CH2)nCH3 I, wherein AA stands for Glu or Lys. In saturated fatty chain alcohol, n is equal to 6, 8, 10, 12, 14 or 16. the invention also relates to a preparation method and application of the conjugates as an immunosuppressant. An experimental result of the saturated fatty chain alcohol His-Gly-AA tripeptide ester which has the inhibition effects of splenic lymphocyte mitogen breeder reaction and macrophage phagocytosis activity indicates that the conjugates of the invention have excellent immunosuppressive action and can be clinically used as an immunosuppressant.

Description

Saturated fatty chain alcohol His-Gly-AA tripeptide ester and synthetic method thereof and application
Technical field
The present invention relates to the saturated aliphatic chain alcohol tripeptide ester, relate in particular to a series of saturated fatty chain alcohol His-Gly-AA tripeptide esters with immunosuppressive activity, also relate to the preparation method of these saturated aliphatic chain alcohol tripeptide esters and they as the application of immunosuppressor, belong to biomedicine field.
Background technology
According to statistics, by the end of the year 2002, various organ transplantations 935792 examples time are carried out in the whole world altogether, wherein renal transplantation 585877 examples time, liver transplantation 112153 examples time, heart transplantation 66559 examples time.In addition, many internal organs combined transplantation such as organ transplantation such as pancreas, lung, small intestine and the heart-lung, pancreas-kidney, liver-kidney, liver-intestines is also all succeedd and is applied to clinical.At present, 1 year people/kidney survival rate of renal transplantation reaches 90%～95%, and 5 annual survival rates surpass 70%.Organ transplantation will form transplantation tolerance, and this just requires the patient to take immunosuppressor all the life.The progress of organ transplantation depends on the progress of immunosuppressor to a great extent.Nearly decades, though new immunosuppressive drug makes organ transplantation get the development of advancing by leaps and bounds clinical, their toxic side effect, for example renal toxicity and bone marrow inhibition remain organ transplantation and make the serious problems that must face.
Cyclosporin A is present clinical immunosuppressor commonly used.Because the water-soluble extreme difference and the renal toxicity of cyclosporin A are very strong, so be that formulation or curative effect are all unsatisfactory.Improve the water-soluble of cyclosporin A preparation, reduce the renal toxicity of cyclosporin A, be the focus of cyclosporin A research always.The urotoxin peptide has immunosuppressive activity.The contriver recognizes that the ester that help a small child urinate by holding his legs apart toxin peptide and fatty chain alcohol are puted together generation has the self-assembly performance, thereby can be as the medicine carrying material with immunosuppressive activity.For example can be used for wrapping up cyclosporin A, reach the dual purpose that improves the water-soluble of cyclosporin A and reduce the cyclosporin A renal toxicity.
Summary of the invention
One of purpose of the present invention is, urotoxin tripeptides and fatty chain alcohol are puted together, and obtains having the saturated aliphatic chain alcohol tripeptide ester conjugate of immunosuppressive activity.
One of purpose of the present invention is achieved through the following technical solutions:
12 kinds of saturated fatty chain alcohol His-Gly-AA tripeptide esters of general formula I with immunosuppressive activity
His-Gly-AA-O-CH 2-(CH 2)nCH 3 I
Wherein, the AA in the general formula I is Glu or Lys, and n is 6,8,10,12,14 or 16.
Two of purpose of the present invention is that a kind of above-mentioned method with saturated fatty chain alcohol His-Gly-AA tripeptide ester of immunosuppressive activity for preparing is provided.
Two of purpose of the present invention is achieved through the following technical solutions:
A kind of 12 kinds of methods that prepare general formula I with saturated fatty chain alcohol His-Gly-AA tripeptide ester of immunosuppressive activity, this method comprises:
(1) according to existing liquid phase synthetic technology; saturated fatty alcohol is progressively connect saturated fatty alcohol three peptide conjugates of the synthetic protecting group protection of peptide with the protection intermediate of L-glutamic acid or Methionin, the protection intermediate of glycine, the protection intermediate of Histidine successively, and described saturated fatty alcohol is CH 3(CH 2) nCH 2OH, n=6,8,10,12,14 or 16;
(2) N end protecting group and the C that sloughs saturated fatty alcohol three peptide conjugates of protecting group protection successively holds protecting group to obtain target compound.
Wherein said N end protecting group is a blocking group commonly used when the N end of polypeptide is protected, and for example can be tertbutyloxycarbonyl (Boc), carbobenzoxy-(Cbz) (Z); Described C end protecting group is a blocking group commonly used when the C end of polypeptide is protected, and for example can be benzyloxy (OBzl); The process of described liquid phase synthetic technology and described protection, deprotection is the conventional and technique known of this area.
When AA was Glu, the preparation method of described saturated fatty alcohol three peptide conjugates can summarize with the route of Fig. 1, and concrete, described method comprises:
(1) at (Boc) 2O and NaOH are converted into the N-t-butoxycarbonyl glycine with glycine under existing;
(2) at (Boc) 2O and triethylamine are converted into N-tertbutyloxycarbonyl-N with Histidine under existing Im-tertbutyloxycarbonyl-Histidine;
(3) in the presence of DCC, HOBt, anhydrous THF with N-tertbutyloxycarbonyl-L-glutamic acid-γ-benzyl ester and saturated fatty alcohol condensation, generate saturated fatty alcohol N-tertbutyloxycarbonyl-γ-benzyl ester-glutamate;
(4) in hydrogenchloride-ethyl acetate, saturated fatty alcohol N-tertbutyloxycarbonyl-γ-benzyl ester-glutamate is removed the tertiary butyloxycarbonyl protecting group, generate saturated fatty alcohol γ-benzyl ester-glutamate;
(5) in anhydrous THF, in the presence of DCC and HOBt,, generate saturated fatty alcohol N-tertbutyloxycarbonyl glycyl γ-benzyl ester glutamate with saturated fatty alcohol γ-benzyl ester-glutamate and the condensation of N-t-butoxycarbonyl glycine;
(6) in the presence of hydrogenchloride-ethyl acetate, saturated fatty alcohol N-tertbutyloxycarbonyl glycyl γ-benzyl ester glutamate is removed tertbutyloxycarbonyl and generate saturated fatty alcohol glycyl γ-benzyl ester-glutamate;
(7) in anhydrous THF, in the presence of DCC and HOBt with saturated fatty alcohol glycyl γ-benzyl ester glutamate and N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl-Histidine condensation generates saturated fatty alcohol N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl histidyl-glycyl γ-benzyl ester glutamate;
(8) in hydrogenchloride-ethyl acetate with saturated fatty alcohol N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl histidyl-glycyl γ-benzyl ester-glutamate removes tertbutyloxycarbonyl, generates saturated fatty alcohol histidyl-glycyl γ-benzyl ester-glutamate;
(9) in dehydrated alcohol, in the presence of Pd/C,, generate saturated fatty alcohol histidyl-glycyl glutamate with saturated fatty alcohol histidyl-glycyl γ-benzyl ester-glutamate hydrogenolysis.
When AA was Lys, the preparation method of described saturated fatty alcohol three peptide conjugates can summarize with the route of Fig. 2, and concrete, described method comprises:
(1) at (Boc) 2O and NaOH are converted into the N-t-butoxycarbonyl glycine with glycine under existing;
(2) at (Boc) 2O and triethylamine are converted into N-tertbutyloxycarbonyl-N with Histidine under existing Im-tertbutyloxycarbonyl-Histidine;
(3) in the presence of DCC, HOBt, anhydrous THF with N α-tertbutyloxycarbonyl-N ε-carbobenzoxy-(Cbz) Methionin and saturated fatty alcohol condensation generate saturated fatty alcohol N α-tertbutyloxycarbonyl-N ε-carbobenzoxy-(Cbz) Methionin ester;
(4) in hydrogenchloride-ethyl acetate with saturated fatty alcohol N α-tertbutyloxycarbonyl-N ε-carbobenzoxy-(Cbz) Methionin ester removes the tertiary butyloxycarbonyl protecting group, generates saturated fatty alcohol N ε-carbobenzoxy-(Cbz) Methionin ester;
(5) in anhydrous THF, in the presence of DCC and HOBt with saturated fatty alcohol N ε-carbobenzoxy-(Cbz) Methionin ester and the condensation of N-t-butoxycarbonyl glycine generate saturated fatty alcohol N-tertbutyloxycarbonyl glycyl N ε-carbobenzoxy-(Cbz) Methionin ester;
(6) in the presence of hydrogenchloride-ethyl acetate with saturated fatty alcohol N-tertbutyloxycarbonyl glycyl N ε-carbobenzoxy-(Cbz) Methionin ester removes tertbutyloxycarbonyl and generates saturated fatty alcohol glycyl N ε-carbobenzoxy-(Cbz) Methionin ester;
(7) in anhydrous THF, in the presence of DCC and HOBt with saturated fatty alcohol glycyl N ε-carbobenzoxy-(Cbz) Methionin ester and N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl-Histidine condensation generates saturated fatty alcohol N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl histidyl-glycyl N ε-carbobenzoxy-(Cbz) Methionin ester;
(8) in hydrogenchloride-ethyl acetate with saturated fatty alcohol N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl histidyl-glycyl N ε-carbobenzoxy-(Cbz) Methionin ester removes tertbutyloxycarbonyl, generates saturated fatty alcohol histidyl-glycyl N ε-carbobenzoxy-(Cbz) Methionin ester;
(9) in dehydrated alcohol, in the presence of Pd/C with saturated fatty alcohol histidyl-glycyl N ε-carbobenzoxy-(Cbz) Methionin ester hydrogenolysis generates saturated fatty alcohol histidyl-glycyl Methionin ester.
Experimental result shows that 12 kinds of saturated fatty chain alcohol His-Gly-AA tripeptide esters with immunosuppressive activity of the present invention have outstanding immunosuppressive action, can be used as immunosuppressor clinically and uses.And equal energy self-assembly granulating is through being stabilized in the nanometer ball of 102-454nm in the saturated fatty chain alcohol His-Gly-AA tripeptide ester aqueous solution of the present invention, can be used as the preparation material of preparation micro emulsion or lipidosome drug carrier, can be used as the targeting preparation material of preparation micro emulsion, lipidosome drug carrier in addition.
Description of drawings
Fig. 1 is 6 kinds of synthetic routes with saturated aliphatic chain alcohol His-Gly-Glu tripeptide ester of immunosuppressive activity when AA in the general formula I is Glu.I) anhydrous THF, DCC, HOBt and NMM; Ii) 4N hydrogenchloride-ethyl acetate solution; Iii) dehydrated alcohol, Pd/C and H 27a n=6,7b n=8,7c n=10,7d n=12,7e n=14,7f n=16.
Fig. 2 is 6 kinds of synthetic routes with saturated aliphatic chain alcohol His-Gly-Lys tripeptide ester of immunosuppressive activity when AA in the general formula I is Lys.I) anhydrous THF, DCC, HOBt and NMM; Ii) 4N hydrogenchloride-ethyl acetate solution; Iii) dehydrated alcohol, Pd/C and H 214a n=6,14b n=8,14c n=10,14d n=12,14e n=14,14f n=16.
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and not should be understood to limitation of the present invention.
Embodiment
Embodiment 1 preparation Boc-Glu (OBzl)-OCH 2(CH 2) 6CH 3
1.00g (2.96mmol) Boc-Glu (OBzl) is dissolved in the anhydrous THF of 20ml, and ice bath adds 0.39g (2.96mmol) N-hydroxy benzo triazole (HOBt) down in the solution that obtains, and makes dissolving fully.Add 0.74g (3.55mmol) dicyclohexyl carbonyl diimine (DCC) after 10 minutes.Obtain reaction solution (I).The following 0.39g of ice bath (2.96mmol) Fatty Alcohol(C12-C14 and C12-C18) CH 3(CH 2) 6CH 2OH is suspended among the anhydrous THF of 20ml, adds 1ml N-methylmorpholine (NMM) then, transfers pH 8-9.Stirred 35 minutes, and obtained reaction solution (II).The following reaction solution of ice bath (I) adds in the reaction solution (II), and first ice bath stirs 1h down, stirring at room 12h again, and TLC (chloroform/methanol, 10: 1) shows that Boc-Glu (Z) disappears.Filtering dicyclohexylurea (DCU) (DCU), THF is removed in decompression.Residue 50ml acetic acid ethyl dissolution.The solution that obtains is used saturated NaHCO successively 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed, 5%KHSO 4The aqueous solution is washed with the saturated NaCl aqueous solution and is washed.The organic phase anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, obtain 1.26g (95%) title compound, are colorless oil.ESI-MS(m/z)：450[M+H] +，[α] 20 D＝-8.4(c＝1.0，CH 3OH)。
Embodiment 2 preparation Boc-Glu (OBzl)-OCH 2(CH 2) 8CH 3
According to the method for embodiment 1 by 1.00g (2.96mmol) Boc-Glu (OBzl) and 0.47g (2.96mmol) CH 3(CH 2) 8CH 2OH makes 1.37g (97%) title compound, is colorless oil.ESI-MS(m/z)：478[M+H] +，[α] 20 D＝-11.6(c＝1.0，CH 3OH)。
Embodiment 3 preparation Boc-Glu (OBzl)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 1 by 1.00g (2.96mmol) Boc-Glu (OBzl) and 0.55g (2.96mmol) CH 3(CH 2) 10CH 2OH makes 1.46g (98%) title compound, is colorless oil.ESI-MS(m/z)：506[M+H] +，[α] 20 D＝-11.5(c＝1.0，CH 3OH)。
Embodiment 4 preparation Boc-Glu (OBzl)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 1 by 1.00g (2.96mmol) Boc-Glu (OBzl) and 0.64g (2.96mmol) CH 3(CH 2) 12CH 2OH makes 1.55g (98%) title compound, is colorless oil.ESI-MS(m/z)：534[M+H] +，[α] 20 D＝-16.6(c＝1.0，CH 3OH)。
Embodiment 5 preparation Boc-Glu (OBzl)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 1 by 1.00g (2.96mmol) Boc-Glu (OBzl) and 0.72g (2.96mmol) CH 3(CH 2) 14CH 2OH makes 1.54g (94%) title compound, is colorless oil.ESI-MS(m/z)：562[M+H] +，[α] 20 D＝-13.8(c＝1.0，CH 3OH)。
Embodiment 6 preparation Boc-Glu (OBzl)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 1 by 1.00g (2.92mmol) Boc-Glu (OBzl) and 0.79g (2.92mmol) CH 3(CH 2) 16CH 2OH makes 1.68g (98%) title compound, is colorless oil.ESI-MS(m/z)：590[M+H] +，[α] 20 D＝-30.2(c＝1.0，CH3OH)。
Embodiment 7 preparation HClGlu (OBzl)-OCH 2(CH 2) 6CH 3
With 1.26g (2.81mmol) Boc-Glu (OBzl)-OCH 2(CH 2) 6CH 3Be dissolved in 25ml 4N hydrogenchloride-ethyl acetate solution, stirring at room 2 hours, TLC (chloroform: methyl alcohol, 5: 1) shows Boc-Glu (OBzl)-OCH 2(CH 2) 6CH 3Disappear, concentrating under reduced pressure is removed ethyl acetate, and residue adds a small amount of ether repeatedly and carries out concentrating under reduced pressure to remove de-chlorine hydride.Add a small amount of ether at last residue is ground to form 1.03g (95%) title compound, be beige oily matter.ESI-MS(m/z)：350[M+H] +，[α] 20 D＝11.5(c＝1.0，CH 3OH)。
Embodiment 8 preparation HClGlu (OBzl)-OCH 2(CH 2) 8CH 3
According to the method for embodiment 7 from 1.37g (2.87mmol) Boc-Glu (OBzl)-OCH 2(CH 2) 8CH 3Make 1.16g (98%) title compound, be beige oily matter.ESI-MS(m/z)：378[M+H] +，[α] 20 D＝21.3(c＝1.0，CH 3OH)。
Embodiment 9 preparation HClGlu (OBzl)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 7, from 1.46g (2.89mmol) Boc-Glu (OBzl)-OCH 2(CH 2) 10CH 3Make 1.15g (90%) title compound, be beige oily matter.ESI-MS(m/z)：406[M+H] +，[α] 20 D＝13.2(c＝1.0，CH 3OH)。
Embodiment 10 preparation HClGlu (OBzl)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 7 from 1.55g (2.91mmol) Boc-Glu (OBzl)-OCH 2(CH 2) 12CH 3Make 1.22g (89%) title compound, be beige oily matter.ESI-MS(m/z)：434[M+H] +，[α] 20 D＝9.5(c＝1.0，CH 3OH)。
Embodiment 11 preparation HClGlu (OBzl)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 7, from 1.54g (2.75mmol) Boc-Glu (OBzl)-OCH 2(CH 2) 14CH 3Make 1.26g (92%) title compound, be colorless solid.ESI-MS(m/z)：462[M+H] +，[α] 20 D＝8.1(c＝1.0，CH 3OH)。
Embodiment 12 preparation HClGlu (OBzl)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 7, from 1.68g (2.85mmol) Boc-Glu (OBzl)-OCH 2(CH 2) 16CH 3Make 1.31g (92%) title compound, be colorless solid.ESI-MS(m/z)：490[M+H] +，[α] 20 D＝13.2(c＝1.0，CH 3OH)。
Embodiment 13 preparation Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 6CH 3
According to the method for embodiment 1 by 0.57g (3.26mmol) Boc-Gly and 1.26g (3.26mmol) HClGlu (OBzl)-OCH 2(CH 2) 6CH 3Make 1.45g (88%) title compound, be faint yellow oily thing.ESI-MS(m/z)：507[M+H] +，[α] 20 D＝-6.4(c＝1.0，CH 3OH)。
Embodiment 14 preparation Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 8CH 3
According to the method for embodiment 1 by 0.58g (3.31mmol) Boc-Gly and 1.37g (3.31mmol) HClGlu (OBzl)-OCH 2(CH 2) 8CH 3Make 1.45g (82%) title compound, be faint yellow oily thing.ESI-MS(m/z)：535[M+H] +，[α] 20 D＝-4.6(c＝1.0，CH 3OH)。
Embodiment 15 preparation Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 1 by 0.45g (2.59mmol) Boc-Gly and 1.15g (2.59mmol) HClGlu (OBzl)-OCH 2(CH 2) 10CH 3Make 1.36g (94%) title compound, be colorless oil.ESI-MS(m/z)：563[M+H] +，[α] 20 D＝-16.4(c＝1.0，CH 3OH)。
Embodiment 16 preparation Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 1 by 0.45g (2.59mmol) Boc-Gly and 1.22g (2.59mmol) HClGlu (OBzl)-OCH 2(CH 2) 12CH 3Make 1.24g (81%) title compound, be faint yellow oily thing.ESI-MS(m/z)：591[M+H] +，[α] 20 D＝-7.1(c＝1.0，CH 3OH)。
Embodiment 17 preparation Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 1 by 0.45g (2.53mmol) Boc-Gly and 1.26g (2.53mmol) HClGlu (OBzl)-OCH 2(CH 2) 14CH 3Make 1.41g (90%) title compound, be faint yellow solid.ESI-MS(m/z)：619[M+H] +，[α] 20 D＝-5.6(c＝1.0，CH 3OH)。
Embodiment 18 preparation Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 1 by 0.47g (2.68mmol) Boc-Gly and 1.41g (2.68mmol) HClGlu (OBzl)-OCH 2(CH 2) 16CH 3Make 1.51g (87%) title compound, be faint yellow solid.ESI-MS(m/z)：647[M+H] +，[α] 20 D＝-5.1(c＝1.0，CH 3OH)。
Embodiment 19 preparation HClGly-Glu (OBzl)-OCH 2(CH 2) 6CH 3
According to the method for embodiment 7, from 1.45g (2.87mmol) Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 6CH 3Make 1.18g (93%) title compound, be beige oily matter.ESI-MS(m/z)：407[M+H] +，[α] 20 D＝-2.8(c＝1.0，CH 3OH)。
Embodiment 20 preparation HClGly-Glu (OBzl)-OCH 2(CH 2) 8CH 3
According to the method for embodiment 7, from 1.45g (2.71mmol) Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 8CH 3Make 1.13g (89%) title compound, be beige oily matter.ESI-MS(m/z)：435[M+H] +，[α] 20 D＝-8.7(c＝1.0，CH 3OH)。
Embodiment 21 preparation HClGly-Glu (OBzl)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 7, from 1.36g (2.42mmol) Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 10CH 3Make 1.16g (96%) title compound, be beige oily matter.ESI-MS(m/z)：463[M+H] +，[α] 20 D＝-6.5(c＝1.0，CH 3OH)。
Embodiment 22 preparation HClGly-Glu (OBzl)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 7, by 1.24g (2.10mmol) Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 12CH 3Make 1.07g (97%) title compound, be beige oily matter.ESI-MS(m/z)：491[M+H] +，[α] 20 D＝-2.8(c＝1.0，CH 3OH)。
Embodiment 23 preparation HClGly-Glu (OBzl)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 7, from 1.41g (2.28mmol) Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 14CH 3Make 1.20g (95%) title compound, be the beige solid.ESI-MS(m/z)：519[M+H] +，[α] 20 D＝-5.8(c＝1.0，CH 3OH)。
Embodiment 24 preparation HClGly-Glu (OBzl)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 7, from 1.51g (2.33mmol) Boc-Gly-Glu (OBzl)-OCH 2(CH 2) 16CH 3Make 1.28g (94%) title compound, be the beige solid.ESI-MS(m/z)：547[M+H] +，[α] 20 D＝-2.1(c＝1.0，CH 3OH)。
Embodiment 25 preparation Boc-His (Boc)-Gly-Glu (OBzl)-OCH 2(CH 2) 6CH 3
According to the method for embodiment 1 by 0.80g (2.25mmol) Boc-His (Boc) and 1.00g (2.25mmol) HClGly-Glu (OBzl)-OCH 2(CH 2) 6CH 3Make, the gained compound gets water white transparency oily thing 1.09g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 65%.ESI-MS(m/z)：744[M+H] +，[α] 20 D＝-14.3(c＝1.0，CH 3OH)。
Embodiment 26 preparation Boc-His (Boc)-Gly-Glu (OBzl)-OCH 2(CH 2) 8CH 3
Method 0.76g (2.12mmol) Boc-His (Boc) and 1.00g (2.12mmol) HClGly-Glu (OBzl)-OCH according to embodiment 1 2(CH 2) 8CH 3Make, the gained compound gets water white transparency oily thing 1.16g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 71%.ESI-MS(m/z)：772[M+H] +，[α] 20 D＝-20.0(c＝1.0，CH 3OH)。
Embodiment 27 preparation Boc-His (Boc)-Gly-Glu (OBzl)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 1 by 0.71g (2.00mmol) Boc-His (Boc) and 1.00g (2.00mmol) HClGly-Glu (OBzl)-OCH 2(CH 2) 10CH 3Make, the gained compound gets water white transparency oily thing 1.09g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 68%.ESI-MS(m/z)：800[M+H] +，[α] 20 D＝-25.8(c＝1.0，CH 3OH)。
Embodiment 28 preparation Boc-His (Boc)-Gly-Glu (OBzl)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 1 by 0.67g (1.89mmol) Boc-His (Boc) and 1.00g (1.89mmol) HClGly-Glu (OBzl)-OCH 2(CH 2) 12CH 3Make, the gained compound gets water white transparency oily thing 1.16g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 74%.ESI-MS(m/z)：828[M+H] +，[α] 20 D＝-19.8(c＝1.0，CH 3OH)。
Embodiment 29 preparation Boc-His (Boc)-Gly-Glu (OBzl)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 1 by 0.71g (1.98mmol) Boc-His (Boc) and 1.10g (1.98mmol) HClGly-Glu (OBzl)-OCH 2(CH 2) 14CH 3Make, the gained compound gets water white transparency oily thing 1.05g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 62%.ESI-MS(m/z)：856[M+H] +，[α] 20 D＝-19.7(c＝1.0，CH 3OH)。
Embodiment 30 preparation Boc-His (Boc)-Gly-Glu (OBzl)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 1 by 0.61g (1.72mmol) Boc-His (Boc) and 1.10g (1.72mmol) HClGly-Glu (OBzl)-OCH 2(CH 2) 16CH 3Make, the gained compound gets water white transparency oily thing 1.08g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 71%.ESI-MS(m/z)：884[M+H] +，[α]20D＝-19.1(c＝1.0，CH 3OH)。
Embodiment 31 preparation HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 6CH 3
According to the method for embodiment 7, from 900mg (1.21mmol) Boc-His (Boc)-Gly-Glu (OBzl)-OCH 2(CH 2) 6CH 3Make 0.65g (92%) title compound, be colorless solid.ESI-MS(m/z)：544[M+H] +。
Embodiment 32 preparation HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 8CH 3
According to the method for embodiment 7, from 900mg (1.16mmol) Boc-His (Boc)-Gly-Glu (OBzl)-OCH 2(CH 2) 8CH 3Make 0.64g (90%) title compound, be colorless solid.ESI-MS(m/z)：572[M+H] +。
Embodiment 33 preparation HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 7, from 850mg (1.06mmol) Boc-His (Boc)-Gly-Glu (OBzl)-OCH 2(CH 2) 10CH 3Make 0.61g (91%) title compound, be colorless solid.ESI-MS(m/z)：600[M+H] +。
Embodiment 34 preparation HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 7,, from 900mg (1.09mmol) Boc-His (Boc)-Gly-Glu (OBzl)-OCH 2(CH 2) 12CH 3Make 0.67g (93%) title compound, be colorless solid.ESI-MS(m/z)：628[M+H] +。
Embodiment 35 preparation HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 7, from 850mg (1.00mmol) Boc-His (Boc)-Gly-Glu (OBzl)-OCH 2(CH 2) 14CH 3Make 0.64g (92%) title compound, be colorless solid.ESI-MS(m/z)：699[M+H] +。
Embodiment 36 preparation HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 7, from 800mg (0.91mmol) Boc-His (Boc)-Gly-Glu (OBOBzll)-OCH 2(CH 2) 16CH 3Make 0.59g (90%) title compound, be colorless solid.ESI-MS(m/z)：684[M+H] +。
Embodiment 37 preparation His-Gly-Glu-OCH 2(CH 2) 6CH 3(7a)
With 650mg (1.11mmol) HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 6CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba), to HClHis-Gly-Glu (OBzL)-OCH 2(CH 2) 6CH 3Disappear.Filtering Pd/C, filtrate decompression are concentrated into drying 447mg (82%) title compound that gets, and are colorless oil.ESI-MS(m/z)：451[M-H] -，[α] 20 D＝-9.8(c＝1.0，CH 3OH)。M.p.：112.2-113.1℃。
Embodiment 38 preparation His-Gly-Glu-OCH 2(CH 2) 8CH 3(7b)
With 640mg (1.04mmol) HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 8CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba) disappear to raw material point.Filtering Pd/C, filtrate decompression are concentrated into drying 445mg (80%) title compound that gets, and are colorless oil.ESI-MS(m/z)：479[M-H] -，[α] 20 D＝-18.7(c＝1.0，CH 3OH)，M.p.：112.9-113.6℃。
Embodiment 39 preparation His-Gly-Glu-OCH 2(CH 2) 10CH 3(7c)
With 610mg (0.96mmol) HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 10CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba) disappear to raw material point.Filtering Pd/C, filtrate decompression are concentrated into drying 448mg (83%) title compound that gets, and are colorless oil.ESI-MS(m/z)：507[M-H] -，[α] 20 D＝-6.6(c＝1.0，CH 3OH)，M.p.：129.7-130.6℃。
Embodiment 40 preparation His-Gly-Glu-OCH 2(CH 2) 12CH 3(7d)
With 670mg (1.01mmol) HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 12CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba) disappear to raw material point.Filtering Pd/C, filtrate decompression are concentrated into drying 437mg (75%) title compound that gets, and are colorless oil.ESI-MS(m/z)：535[M-H] -，[α] 20 D＝-5.7(c＝1.0，CH 3OH)，M.p.：88.1-88.9℃。
Embodiment 41 preparation His-Gly-Glu-OCH 2(CH 2) 14CH 3(7e)
With 640mg (0.92mmol) HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 14CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba) disappear to raw material point.Filtering Pd/C, filtrate decompression are concentrated into drying 473mg (84%) title compound that gets, and are colorless oil.ESI-MS(m/z)：563[M-H] -，[α] 20 D＝-1.9(c＝1.0，CH 3OH)，M.p.：124.0-125.9℃。
Embodiment 42 preparation His-Gly-Glu-OCH 2(CH 2) 16CH 3(7f)
With 590mg (0.82mmol) HClHis-Gly-Glu (OBzl)-OCH 2(CH 2) 16CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba) disappear to raw material point.Filtering Pd/C, filtrate decompression are concentrated into drying 385mg (72%) title compound that gets, and are colorless oil.ESI-MS(m/z)：591[M-H] -，[α] 20 D＝-5.1(c＝1.0，CH 3OH)，M.p.：84.1-85.2℃。
Embodiment 43 preparation Boc-Lys (Z)-OCH 2(CH 2) 6CH 3
1.00g (2.6mmol) Boc-Lys (Z) is dissolved in the anhydrous THF of 20ml, and ice bath adds 0.35g (2.6mmol) N-hydroxy benzo triazole (HOBt) down in the solution that obtains, and makes dissolving fully.Add 0.65g (3.1mmol) dicyclohexyl carbonyl diimine (DCC) after 10 minutes.Obtain reaction solution (III).The following 0.35g of ice bath (2.6mmol) Fatty Alcohol(C12-C14 and C12-C18) CH 3(CH 2) 6CH 2OH is suspended among the anhydrous THF of 20ml, adds 1ml N-methylmorpholine (NMM) then, transfers pH 8-9.Stirred 35 minutes, and obtained reaction solution (IV).The following reaction solution of ice bath (III) adds in the reaction solution (IV), and first ice bath stirs 1h down, stirring at room 12h again, and TLC (chloroform/methanol, 10: 1) shows that Boc-Lys (Z) disappears.Filtering dicyclohexylurea (DCU) (DCU), THF is removed in decompression.Residue 50ml acetic acid ethyl dissolution.The solution that obtains is used saturated NaHCO successively 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed, 5%KHSO 4The aqueous solution is washed with the saturated NaCl aqueous solution and is washed.The organic phase anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, obtain 1.25g (98%) title compound, are colorless oil.ESI-MS(m/z)：493[M+H] +，[α] 20 D＝-21.0(c＝1.0，CH 3OH)。
Embodiment 44 preparation Boc-Lys (Z)-OCH 2(CH 2) 8CH 3
According to the method for embodiment 43 by 1.00g (2.6mmol) Boc-Lys (Z) and 0.42g (2.6mmol) CH 3(CH 2) 8CH 2OH makes 1.20g (89%) title compound, is colorless oil.ESI-MS(m/z)：521[M+H] +，[α] 20 D＝-20.9(c＝1.0，CH 3OH)。
Embodiment 45 preparation Boc-Lys (Z)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 43 by 1.00g (2.6mmol) Boc-Lys (Z) and 0.48g (2.6mmol) CH 3(CH 2) 10CH 2OH makes 1.31g (92%) title compound, is colorless oil.ESI-MS(m/z)：549[M+H] +，[α] 20 D＝-14.4(c＝1.0，CH 3OH)。
Embodiment 46 preparation Boc-Lys (Z)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 43 by 1.00g (2.6mmol) Boc-Lys (Z) and 0.55g (2.6mmol) CH 3(CH 2) 12CH 2OH makes 1.35g (93%) title compound, is colorless oil.ESI-MS(m/z)：577[M+H] +，[α] 20 D＝-8.9(c＝1.0，CH 3OH)。
Embodiment 47 preparation Boc-Lys (Z)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 43 by 1.00g (2.6mmol) Boc-Lys (Z) and 0.63g (2.6mmol) CH 3(CH 2) 14CH 2OH makes 1.55g (99%) title compound, is colorless oil.ESI-MS(m/z)：604[M+H] +，[α] 20 D＝-11.9(c＝1.0，CH 3OH)。
Embodiment 48 preparation Boc-Lys (Z)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 43 by 1.00g (2.6mmol) Boc-Lys (Z) and 0.69g (2.6mmol) CH 3(CH 2) 16CH 2OH makes 1.56g (95%) title compound, is colorless oil.ESI-MS(m/z)：632[M+H] +，[α] 20 D＝-30.2(c＝1.0，CH 3OH)。
Embodiment 49 preparation HClLys (Z)-OCH 2(CH 2) 6CH 3
With 1.25g (2.54mmol) Boc-Lys (Z)-OCH 2(CH 2) 6CH 3Be dissolved in 25ml 4N hydrogenchloride-ethyl acetate solution, stirring at room 2 hours, TLC (chloroform: methyl alcohol, 5: 1) shows Boc-Lys (Z)-OCH 2(CH 2) 6CH 3Disappear, concentrating under reduced pressure is removed ethyl acetate, and residue adds a small amount of ether repeatedly and carries out concentrating under reduced pressure to remove de-chlorine hydride.Add a small amount of ether at last residue is ground to form 1.03g (95%) title compound, be beige oily matter.ESI-MS(m/z)：393[M+H] +，[α] 20 D＝100.3(c＝1.0，CH 3OH)。
Embodiment 50 preparation HClLys (Z)-OCH 2(CH 2) 8CH 3
According to the method for embodiment 49, from 1.20g (2.31mmol) Boc-Lys (Z)-OCH 2(CH 2) 8CH 3Make 0.95g (90%) title compound, be beige oily matter.ESI-MS(m/z)：421[M+H] +，[α] 20 D＝91.3(c＝1.0，CH 3OH)。
Embodiment 51 preparation HClLys (Z)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 49, from 1.31g (2.39mmol) Boc-Lys (Z)-OCH 2(CH 2) 10CH 3Make 1.07g (92%) title compound, be beige oily matter.ESI-MS(m/z)：449[M+H] +，[α] 20 D＝78.7(c＝1.0，CH 3OH)。
Embodiment 52 preparation HClLys (Z)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 49, from 1.35g (2.34mmol) Boc-Lys (Z)-OCH 2(CH 2) 12CH 3Make 0.99g (89%) title compound, be beige oily matter.ESI-MS(m/z)：477[M+H] +，[α] 20 D＝79.0(c＝1.0，CH 3OH)。
Embodiment 53 preparation HClLys (Z)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 49, from 1.55g (2.57mmol) Boc-Lys (Z)-OCH 2(CH 2) 14CH 3Make 1.36g (98%) title compound, be colorless solid.ESI-MS(m/z)：505[M+H] +，[α] 20 D＝71.3(c＝1.0，CH 3OH)。
Embodiment 54 preparation HClLys (Z)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 49, from 1.56g (2.47mmol) Boc-Lys (Z)-OCH 2(CH 2) 16CH 3Make 1.23g (88%) title compound, be colorless solid.ESI-MS(m/z)：533[M+H] +，[α] 20 D＝65.0(c＝1.0，CH 3OH)。
Embodiment 55 preparation Boc-Gly-Lys (Z)-OCH 2(CH 2) 6CH 3
According to the method for embodiment 43 by 0.41g (2.33mmol) Boc-Gly and 1.00g (2.33mmol) HClLys (Z)-OCH 2(CH 2) 6CH 3Make 1.25g (98%) title compound, be colorless oil.ESI-MS(m/z)：550[M+H] +，[α] 20 D＝-9.7(c＝1.0，CH 3OH)。
Embodiment 56 preparation Boc-Gly-Lys (Z)-OCH 2(CH 2) 8CH 3
According to the method for embodiment 43 by 0.35g (1.97mmol) Boc-Gly and 0.90g (1.97mmol) HClLys (Z)-OCH 2(CH 2) 8CH 3Make 1.08g (95%) title compound, be colorless oil.ESI-MS(m/z)：578[M+H] +，[α] 20 D＝-1.8(c＝1.0，CH 3OH)。
Embodiment 57 preparation Boc-Gly-Lys (Z)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 43 by 0.33g (1.85mmol) Boc-Gly and 0.90g (1.85mmol) HClLys (Z)-OCH 2(CH 2) 10CH 3Make 1.11g (99%) title compound, be colorless oil.ESI-MS(m/z)：606[M+H] +，[α] 20 D＝-2.2(c＝1.0，CH 3OH)。
Embodiment 58 preparation Boc-Gly-Lys (Z)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 43 by 0.38g (2.15mmol) Boc-Gly and 1.10g (2.15mmol) HClLys (Z)-OCH 2(CH 2) 12CH 3Make 1.28g (94%) title compound, be colorless oil.ESI-MS(m/z)：634[M+H] +，[α] 20 D＝-0.9(c＝1.0，CH 3OH)。
Embodiment 59 preparation Boc-Gly-Lys (Z)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 43 by 0.36g (2.04mmol) Boc-Gly and 1.10g (2.04mmol) HClLys (Z)-OCH 2(CH 2) 14CH 3Make 1.31g (97%) title compound, be colorless oil.ESI-MS(m/z)：662[M+H] +，[α] 20 D＝-8.4(c＝1.0，CH 3OH)。
Embodiment 60 preparation Boc-Gly-Lys (Z)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 43 by 0.28g (1.59mmol) Boc-Gly and 1.10g (1.59mmol) HClLys (Z)-OCH 2(CH 2) 16CH 3Make 1.08g (99%) title compound, be colorless oil.ESI-MS(m/z)：690[M+H] +，[α] 20 D＝-6.5(c＝1.0，CH 3OH)。
Embodiment 61 preparation HClGly-Lys (Z)-OCH 2(CH 2) 6CH 3
According to the method for embodiment 49, from 1.20g (2.18mmol) Boc-Gly-Lys (Z)-OCH 2(CH 2) 6CH 3Make 0.85g (80%) title compound, be beige oily matter.ESI-MS(m/z)：450[M+H] +，[α] 20 D＝-19.0(c＝1.0，CH 3OH)。
Embodiment 62 preparation HClGly-Lys (Z)-OCH 2(CH 2) 8CH 3
According to the method for embodiment 49, from 1.00g (1.73mmol) Boc-Gly-Lys (Z)-OCH 2(CH 2) 8CH 3Make 0.76g (85%) title compound, be beige oily matter.ESI-MS(m/z)：478[M+H] +，[α] 20 D＝-47.7(c＝1.0，CH 3OH)。
Embodiment 63 preparation HClGly-Lys (Z)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 49, from 1.00g (1.65mmol) Boc-Gly-Lys (Z)-OCH 2(CH 2) 10CH 3Make 0.80g (89%) title compound, be beige oily matter.ESI-MS(m/z)：506[M+H] +，[α] 20 D＝-94.3(c＝1.0，CH 3OH)。
Embodiment 64 preparation HClGly-Lys (Z)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 49, from 1.10g (1.74mmol) Boc-Gly-Lys (Z)-OCH 2(CH 2) 12CH 3Make 0.80g (81%) title compound, be beige oily matter.ESI-MS(m/z)：534[M+H] +，[α] 20 D＝-53.0(c＝1.0，CH 3OH)。
Embodiment 65 preparation HClGly-Lys (Z)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 49, from 1.20g (1.82mmol) Boc-Gly-Lys (Z)-OCH 2(CH 2) 14CH 3Make 0.98g (91%) title compound, be the beige solid.ESI-MS(m/z)：562[M+H] +，[α] 20 D＝-17.7(c＝1.0，CH 3OH)。
Embodiment 66 preparation HClGly-Lys (Z)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 49, from 1.00g (1.45mmol) Boc-Gly-Lys (Z)-OCH 2(CH 2) 16CH 3Make 0.85g (94%) title compound, be the beige solid.ESI-MS(m/z)：590[M+H] +，[α] 20 D＝-35.0(c＝1.0，CH 3OH)。
Embodiment 67 preparation Boc-His (Boc)-Gly-Lys (Z)-OCH 2(CH 2) 6CH 3
According to the method for embodiment 43 by 0.62g (1.75mmol) Boc-His (Boc) and 0.85g (1.75mmol) HClGly-Lys (Z)-OCH 2(CH 2) 6CH 3And through silica gel column chromatography (chloroform: methyl alcohol=100: 1) purifying makes 949mg (69%) title compound, is water white transparency oily thing.ESI-MS(m/z)：787[M+H] +，[α] 20 D＝-17.4(c＝1.0，CH 3OH)。
Embodiment 68 preparation Boc-His (Boc)-Gly-Lys (Z)-OCH 2(CH 2) 8CH 3
According to the method for embodiment 43 by 0.53g (1.48mmol) Boc-His (Boc) and 0.76g (1.48mmol) HClGly-Lys (Z)-OCH 2(CH 2) 8CH 3And through silica gel column chromatography (chloroform: methyl alcohol=100: 1) purifying makes 807mg (67%) title compound, is water white transparency oily thing.ESI-MS(m/z)：815[M+H] +，[α] 20 D＝-16.0(c＝1.0，CH 3OH)。
Embodiment 69 preparation Boc-His (Boc)-Gly-Lys (Z)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 43 by 0.53g (1.48mmol) Boc-His (Boc) and 0.80g (1.48mmol) HClGly-Lys (Z)-OCH 2(CH 2) 10CH 3And through silica gel column chromatography (chloroform: methyl alcohol=100: 1) purifying makes 796mg (64%) title compound, is water white transparency oily thing.ESI-MS(m/z)：843[M+H] +，[α] 20 D＝-17.6(c＝1.0，CH 3OH)。
Embodiment 70 preparation Boc-His (Boc)-Gly-Lys (Z)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 43 by 0.50g (1.40mmol) Boc-His (Boc) and 0.80g (1.40mmol) HClGly-Lys (Z)-OCH 2(CH 2) 12CH 3And through silica gel column chromatography (chloroform: methyl alcohol=100: 1) purifying makes 721mg (59%) title compound, is water white transparency oily thing.ESI-MS(m/z)：871[M+H] +，[α] 20 D＝-24.9(c＝1.0，CH 3OH)。
Embodiment 71 preparation Boc-His (Boc)-Gly-Lys (Z)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 43 by 0.58g (1.64mmol) Boc-His (Boc) and 0.98g (1.64mmol) HClGly-Lys (Z)-OCH 2(CH 2) 14CH 3And through silica gel column chromatography (chloroform: methyl alcohol=100: 1) purifying makes 751mg (51%) title compound, is water white transparency oily thing.ESI-MS(m/z)：899[M+H] +，[α] 20 D＝-18.6(c＝1.0，CH 3OH)。
Embodiment 72 preparation Boc-His (Boc)-Gly-Lys (Z)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 43 by 0.45g (1.28mmol) Boc-His (Boc) and 0.80g (1.28mmol) HClGly-Lys (Z)-OCH 2(CH 2) 14CH 3And through silica gel column chromatography (chloroform: methyl alcohol=100: 1) purifying makes 840mg (71%) title compound, is water white transparency oily thing.ESI-MS(m/z)：927[M+H] +，[α] 20 D＝-21.3(c＝1.0，CH 3OH)。
Embodiment 73 preparation HClHis-Gly-Lys (Z)-OCH 2(CH 2) 6CH 3
According to the method for embodiment 49, from 786mg (1.00mmol)
Boc-His (Boc)-Gly-Lys (Z)-NHCH 2(CH 2) 6CH 3Make 0.57g (91%) title compound, be colorless solid.ESI-MS(m/z)：587[M+H] +。
Embodiment 74 preparation HClHis-Gly-Lys (Z)-OCH 2(CH 2) 8CH 3
According to the method for embodiment 49, from 700mg (0.86mmol)
Boc-His (Boc)-Gly-Lys (Z)-NHCH 2(CH 2) 8CH 3Make 0.50g (90%) title compound, be colorless solid.ESI-MS(m/z)：615[M+H] +。
Embodiment 75 preparation HClHis-Gly-Lys (Z)-OCH 2(CH 2) 10CH 3
According to the method for embodiment 49, from 700mg (0.83mmol)
Boc-His (Boc)-Gly-Lys (Z)-NHCH 2(CH 2) 1oCH 3Make 0.52g (92%) title compound, be colorless solid.ESI-MS(m/z)：643[M+H] +。
Embodiment 76 preparation HClHis-Gly-Lys (Z)-OCH 2(CH 2) 12CH 3
According to the method for embodiment 49, from 700mg (0.81mmol)
Boc-His (Boc)-Gly-Lys (Z)-NHCH 2(CH 2) 12CH 3Make 0.53g (93%) title compound, be colorless solid.ESI-MS(m/z)：671[M+H] +。
Embodiment 77 preparation HClHis-Gly-Lys (Z)-OCH 2(CH 2) 14CH 3
According to the method for embodiment 49, from 700mg (0.78mmol)
Boc-His (Boc)-Gly-Lys (Z)-NHCH 2(CH 2) 14CH 3Make 0.54g (95%) title compound, be colorless solid.ESI-MS(m/z)：699[M+H] +。
Embodiment 78 preparation HClHis-Gly-Lys (Z)-OCH 2(CH 2) 16CH 3
According to the method for embodiment 49, from 800mg (0.86mmol)
Boc-His (Boc)-Gly-Lys (Z)-NHCH 2(CH 2) 16CH 3Make 0.59g (90%) title compound, be colorless solid.ESI-MS(m/z)：727[M+H] +。
Embodiment 79 preparation His-Gly-Lys-OCH 2(CH 2) 6CH 3(14a)
With 570mg (0.92mmol) HClHis-Gly-Lys (Z)-OCH 2(CH 2) 6CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba), to HClHis-Gly-Lys (Z)-OCH 2(CH 2) 6CH 3Disappear.Filtering Pd/C, filtrate decompression are concentrated into dried 341mg (77%) title compound, are colorless oil.ESI-MS(m/z)：452[M-H] -，[α] 20 D＝-12.9(c＝1.0，CH 3OH)，M.p.：115.0-116.2℃。
Embodiment 80 preparation His-Gly-Lys-OCH 2(CH 2) 8CH 3(14b)
With 500mg (0.77mmol) HClHis-Gly-Lys (Z)-OCH 2(CH 2) 8CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba) disappear to raw material point.Filtering Pd/C, filtrate decompression concentrate as for getting 376mg (94%) title compound, are colorless oil.ESI-MS(m/z)：480[M-H] -，[α] 20 D＝-14.9(c＝1.0，CH 3OH)，M.p.：139.2-140.5℃。
Embodiment 81 preparation His-Gly-Lys-OCH 2(CH 2) 10CH 3(14c)
With 520mg (0.77mmol) HClHis-Gly-Lys (Z)-OCH 2(CH 2) 10CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba) disappear to raw material point.Filtering Pd/C, filtrate decompression are concentrated into dried 360mg (87%) title compound, are colorless oil.ESI-MS(m/z)：508[M-H] -，[α] 20 D＝-9.3(c＝1.0，CH 3OH)，M.p.：136.5-137.7℃。
Embodiment 82 preparation His-Gly-Lys-OCH 2(CH 2) 12CH 3(14d)
With 530mg (0.75mmol) HClHis-Gly-Lys (Z)-OCH 2(CH 2) 12CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba) disappear to raw material point.Filtering Pd/C, filtrate decompression are concentrated into dried 365mg (85%) title compound, are colorless oil.ESI-MS(m/z)：536[M-H] -，[α] 20 D＝-5.2(c＝1.0，CH 3OH)，M.p.：131.7-132.8℃。
Embodiment 83 preparation His-Gly-Lys-OCH 2(CH 2) 14CH 3(14e)
With 540mg (0.74mmol) HClHis-Gly-Lys (Z)-OCH 2(CH 2) 14CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba) disappear to raw material point.Filtering Pd/C, filtrate decompression are concentrated into dried 360mg (81%) title compound, are colorless oil.ESI-MS(m/z)：564[M-H] -)[α] 20 D＝-6.3(c＝1.0，CH 3OH)，M.p.：136.1-136.7℃。
Embodiment 84 preparation His-Gly-Lys-OCH 2(CH 2) 16CH 3(14f)
With 592mg (0.77mmol) HClHis-Gly-Lys (Z)-OCH 2(CH 2) 16CH 3With dissolve with ethanol, the about 5%Pd/C of adding, stirring at room and logical H 2(0.02Mba) disappear to raw material point.Filtering Pd/C, filtrate decompression are concentrated into dried 379mg (78%) title compound, are colorless oil.ESI-MS(m/z)：592[M-H] -，[α] 20 D＝-5.1(c＝1.0，CH 3OH)，M.p.：137.2-138.5℃。
Test example 1 saturated fatty chain alcohol His-Gly-AA tripeptide ester is to the effect of mouse spleen lymphocyte mitogen inhibition of proliferation
Take off neck and put to death mouse, the aseptic spleen of getting grinds with 200 order steel meshes and piston, use HANK ' S liquid to wash under twice, 1500 rev/min of condition centrifugal 10 minutes, counts then being made into 5 * 10 with complete RPMI-1640 nutrient solution 6/ ml splenic lymphocyte, (every hole contains 5 * 10 in 96 well culture plates to add 100 μ l cell suspensions 6Individual cell).Every hole adds 20 μ l ConA (the ConA final concentration is 5 μ g/ml), and it is 0.05 CO that this 96 well culture plate places volume fraction 2Cultivate 4h for 37 ℃ in the incubator of saturated humidity.Add different concns saturated fatty chain alcohol His-Gly-AA tripeptide ester (1 * 10 behind the 4h respectively -4M, 8 * 10 -5M, 5 * 10 -5M, 3 * 10 -5M, 1 * 10 -5M, 8 * 10 -6M, 5 * 10 -6M and 1 * 10 -6M), 3 multiple holes of each concentration.Establish simultaneously and do not contain the compound control wells and only contain the cell blank hole of not having ConA with the amount nutrient solution.(n=3) all repeated in each hole 3 times.Use the restraining effect of mtt assay detection compound after cultivating 48h to splenic lymphocyte.
Calculate the restraining effect of different concns saturated fatty chain alcohol His-Gly-AA tripeptide ester according to formula " inhibiting rate=(D contrast-D pastille)/D contrast * 100% " to spleen lymphocyte proliferation, draw cell growth curve according to cell relative survival rate and compound concentrations, utilize this growth curve try to achieve the half inhibiting rate ( DxIC 50).The result lists table 1 in.The result shows that saturated fatty chain alcohol His-Gly-AA tripeptide ester of the present invention has clear and definite restraining effect to mice spleen lymphocytes proliferation.
Table 1 saturated fatty chain alcohol His-Gly-AA tripeptide ester is to the effect of mouse spleen lymphocyte mitogen inhibition of proliferation
Figure B2009100853253D0000191
Figure B2009100853253D0000201
Test example 2 saturated fatty chain alcohol His-Gly-AA tripeptide esters are to the restraining effect of macrophage phagocytic
Growth conditions is good, be in the Ana-1 mouse macrophage of logarithmic phase with 1 * 10 5The density of individual/mL is inoculated in 96 orifice plates, every hole 100 μ l, 37 ℃, 5%CO 2Cultivated 4 hours in the incubator, by the saturated fatty chain alcohol His-Gly-AA tripeptide ester (1 * 10 of default concentration gradient adding through sterilising treatment -4M, 8 * 10 -5M, 5 * 10 -5M, 3 * 10 -5M, 1 * 10 -5M, 8 * 10 -6M, 5 * 10 -6M and 1 * 10 -6M), 3 multiple holes of each concentration, control group adds the solvent of isopyknic sample dissolution.Continue to cultivate after 24 hours, inhale and abandon supernatant liquor, every hole adds the neutral red solution of 100 μ l0.1%, places 37 ℃ to hatch 30 minutes.Neutral red solution is abandoned in suction, and washs 2-3 time (removing not by the toluylene red of macrophage phagocytic) with PBS buffered soln, and (ethanol: 100 μ l acetate=1: 1), 4 ℃ are spent the night, microplate reader detection absorbance, wavelength 540nm to add cytolysate.(n=3) all repeated in each hole 3 times.
Calculate the restraining effect of the compound of different concns according to formula " inhibiting rate=(D contrast-D pastille)/D contrast * 100% " to the phagocytic activity of scavenger cell, draw cell growth curve according to cell relative survival rate and compound concentrations, utilize this growth curve try to achieve the half inhibiting rate ( DxIC 50).The result lists table 2 in.The result shows that saturated fatty chain alcohol His-Gly-AA tripeptide ester of the present invention engulfs the Ana-1 mouse macrophage clear and definite restraining effect is arranged.
The restraining effect that table 2 saturated fatty chain alcohol His-Gly-AA tripeptide ester is engulfed the Ana-1 mouse macrophage
Figure B2009100853253D0000211
Test example 3 saturated fatty chain alcohol His-Gly-AA tripeptide esters are in the self-assembly of nanometer level
Saturated fatty chain alcohol His-Gly-AA tripeptide ester is configured to the aqueous solution of 1 μ mol/ml, on the laser nano particle size analyzer, measures particle diameters for 25 ℃.METHOD FOR CONTINUOUS DETERMINATION 8 days writes down its particle diameter.The result lists table 3 in.Data show that equal energy self-assembly granulating is through being stabilized in the nanometer ball of 102-454nm in the saturated fatty chain alcohol His-Gly-AA tripeptide ester aqueous solution of the present invention.
The nanometer ball particle diameter of table 3 saturated fatty chain alcohol His-Gly-AA tripeptide ester self-assembly in water

Claims (5)

1. 12 of general formula I kinds of saturated fatty chain alcohol His-Gly-AA tripeptide esters with immunosuppressive activity:
His-Gly-AA-O-CH 2-(CH 2)nCH 3 I
AA in the general formula I is Glu or Lys, n=6,8,10,12,14 or 16.
2. method for preparing the saturated fatty chain alcohol His-Gly-AA tripeptide ester of claim 1 comprises:
(1) according to existing liquid phase synthetic technology; saturated fatty alcohol is progressively connect saturated fatty alcohol three peptide conjugates of the synthetic protecting group protection of peptide with the protection intermediate of L-glutamic acid or Methionin, the protection intermediate of glycine, the protection intermediate of Histidine successively, and described saturated fatty alcohol is CH 3(CH 2) nCH 2OH, n=6,8,10,12,14 or 16;
(2) N end protecting group and the C that sloughs saturated fatty alcohol three peptide conjugates of protecting group protection successively holds protecting group to obtain target compound.
3. 12 of claim 1 kinds of saturated fatty chain alcohol His-Gly-AA tripeptide ester application in preparation immunosuppressor class medicine.
4. 12 of claim 1 kinds of saturated fatty chain alcohol His-Gly-AA tripeptide esters are as the purposes of the preparation material of preparation micro emulsion or lipidosome drug carrier.
5. 12 of claim 1 kinds of saturated fatty chain alcohol His-Gly-AA tripeptide esters are as the purposes in the targeting preparation material of preparation micro emulsion, lipidosome drug carrier.
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