CN101899087B - Saturated fatty chain amine Glu-Asp-Gly tripeptide amides as well as synthesis method and application thereof - Google Patents

Saturated fatty chain amine Glu-Asp-Gly tripeptide amides as well as synthesis method and application thereof Download PDF

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CN101899087B
CN101899087B CN2009100853234A CN200910085323A CN101899087B CN 101899087 B CN101899087 B CN 101899087B CN 2009100853234 A CN2009100853234 A CN 2009100853234A CN 200910085323 A CN200910085323 A CN 200910085323A CN 101899087 B CN101899087 B CN 101899087B
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saturated fatty
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obzl
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CN101899087A (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 six saturated fatty chain amine Glu-Asp-Gly tripeptide amide conjugates with immunodepression activity, which are represented in the general formula I. In saturated fatty chain amine, n equals to 6, 8, 10, 12, 14 or 16. The invention also relates to a preparation method thereof and an application thereof as immunosuppressant. The depressive action of the saturated fatty chain amine Glu-Asp-Gly tripeptide amides on the proliferation reaction of splenic lymphocyte mitogen and the phagocytic activity of macrophagocyte is tested, and test results show that the compounds have excellent immunodepressive action and can be clinically used as immunosuppressant. The general formula I is represented as Glu-Asp-Gly-NH-CH2-(CH2)nCH3.

Description

Saturated fatty streptamine Glu-Asp-Gly tripeptide amide and compound method and application
Technical field
The present invention relates to saturated fatty streptamine tripeptide amide; Relate in particular to a series of saturated fatty streptamine Glu-Asp-Gly tripeptide amides with immunosuppressive activity; The preparation method and they that also relate to these saturated fatty streptamine tripeptide amides belong to biomedicine field as the application of immunosuppressor.
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 acid amides that help a small child urinate by holding his legs apart toxin peptide and fatty streptamine 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 and improve the water-soluble of cyclosporin A and the dp that reduces the cyclosporin A renal toxicity.
Summary of the invention
One of the object of the invention is, urotoxin tripeptides and fatty streptamine are puted together, and obtains having the saturated fatty streptamine tripeptide amide conjugate of immunosuppressive activity.
One of the object of the invention is realized by the following technical programs:
6 kinds of saturated fatty streptamine Glu-Asp-Gly tripeptide amides of general formula I with immunosuppressive activity
Glu-Asp-Gly-NH-CH 2-(CH 2)nCH 3 I
Wherein, the n=6 in the general formula I, 8,10,12,14 or 16.
Two of the object of the invention is that a kind of above-mentioned method with saturated fatty streptamine Glu-Asp-Gly tripeptide amide of immunosuppressive activity for preparing is provided.
Two of the object of the invention is realized through following technical scheme:
A kind of 6 kinds of preparing general formula I have the method for the saturated fatty streptamine Glu-Asp-Gly tripeptide amide of immunosuppressive activity, and this method comprises:
(1) with the glycocoll and the condensation of saturated fatty amine of the protection of N end protection base, said saturated fatty amine is CH 3(CH 2) nCH 2NH 2, n=6,8,10,12,14 or 16;
(2) slough N end protection base and obtain saturated fatty amine glycocoll acid amides;
(3), the protection midbody of saturated fatty amine glycocoll acid amides and aspartic acid, the protection midbody of L-glutamic acid are progressively connect saturated fatty amine three peptide conjugates of the synthetic protection base of peptide protection according to existing liquid phase synthetic technology;
(4) the N end of sloughing saturated fatty amine three peptide conjugates of protection base protection successively protects base and C end protection base to obtain target compound.
Wherein said N end protection base is the blocking group of using always when the N end of polypeptide is protected, and for example can be tertbutyloxycarbonyl (Boc); Said C end protection base is the blocking group of using always when the C end of polypeptide is protected, and for example can be benzyloxy (OBzl); The process of said liquid phase synthetic technology and said protection, condensation, deprotection is the conventional and technique known of this area.
This preparation method can use the route of Fig. 1 to summarize, and concrete, said method comprises:
(1) at (Boc) 2O and NaOH are converted into the N-t-butoxycarbonyl glycine with glycocoll under existing;
(2) in the presence of DCC, HOBt, anhydrous THF with N-t-butoxycarbonyl glycine and the condensation of saturated fatty amine, generate saturated fatty amine N-t-butoxycarbonyl glycine acid amides;
(3) in hydrogenchloride-ETHYLE ACETATE, saturated fatty amine N-t-butoxycarbonyl glycine acid amides is removed tertiary butyloxycarbonyl protection base, generate saturated fatty amine glycocoll acid amides;
(4) in anhydrous THF, in the presence of DCC and HOBt,, generate saturated fatty amine N-tertbutyloxycarbonyl-β-benzyl ester aspartoyl glycocoll acid amides with saturated fatty amine glycocoll acid amides and N-tertbutyloxycarbonyl-β-benzyl ester aspartic acid condensation;
(5) in the presence of hydrogenchloride-ETHYLE ACETATE, saturated fatty amine N-tertbutyloxycarbonyl-β-benzyl ester aspartoyl glycocoll acid amides is removed tertbutyloxycarbonyl, generate saturated fatty amine β-benzyl ester-aspartoyl glycocoll acid amides;
(6) in anhydrous THF; In the presence of DCC and HOBt,, generate saturated fatty amine N-tertbutyloxycarbonyl-γ-benzyl ester glutamy-β-benzyl ester aspartoyl glycocoll acid amides with saturated fatty amine β-benzyl ester aspartoyl glycocoll acid amides and N-tertbutyloxycarbonyl-γ-benzyl ester L-glutamic acid condensation;
(7) in hydrogenchloride-ETHYLE ACETATE, saturated fatty amine N-tertbutyloxycarbonyl-γ-benzyl ester glutamy β-benzyl ester aspartoyl glycocoll acid amides is removed tertbutyloxycarbonyl, generate saturated fatty amine γ-benzyl ester glutamy β-benzyl ester aspartoyl glycocoll acid amides;
(8) in absolute ethyl alcohol, in the presence of Pd/C,, generate saturated fatty amine glutamy aspartoyl glycocoll acid amides with saturated fatty amine γ-benzyl ester glutamy-β-benzyl ester aspartoyl glycocoll acid amides hydrogenolysis.
Experimental result shows that 6 kinds of saturated fatty streptamine Glu-Asp-Gly tripeptide amides with immunosuppressive activity of the present invention have outstanding immunosuppressive action, can be used as immunosuppressor clinically and uses.And equal ability self-assembly granulating is through being stabilized in the nanometer ball of 38.9-1030nm in the saturated fatty streptamine Glu-Asp-Gly tripeptide amide aqueous solution of the present invention; Can be used as the formulation materials 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 fatty streptamine Glu-Asp-Gly tripeptide amide of immunosuppressive activity of general formula I.I) anhydrous THF, DCC, HOBt and NMM; Ii) 4N hydrogenchloride-ethyl acetate solution; Iii) absolute ethyl alcohol, Pd/C and H 27a n=6,7b n=8,7c n=10,7d n=12,7e n=14,7f 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 are not to be understood that to be limitation of the present invention.
Embodiment
Embodiment 1Boc-Gly-NHCH 2(CH 2) 6CH 3Preparation
0.80g (4.57mmol) Boc-Gly is dissolved in the anhydrous THF of 20ml, and ice bath adds 0.62g (4.57mmol) N-hydroxy benzo triazole (HOBt) down in the solution that obtains, and makes dissolving fully.Add 1.13g (5.48mmol) dicyclohexyl carbonyl diimine (DCC) after 10 minutes, obtain reaction solution (I).The following 0.72g of ice bath (4.57mmol) aliphatic amide CH 3(CH 2) 6CH 2NH 2Be suspended among the anhydrous THF of 20ml, add 1ml N-methylmorpholine (NMM) then, transfer 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-Gly disappears.Filtering NSC 30023 (DCU), THF is removed in decompression.Residue is used the 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.Organic phase is used anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, obtain 1.24g (95%) title compound, are colorless oil.ESI-MS(m/z)：287[M+H] +。
Embodiment 2Boc-Gly-NHCH 2(CH 2) 8CH 3Preparation
According to the method for embodiment 1 by 0.80g (4.57mmol) Boc-Gly and 0.72g (4.57mmol) CH 3(CH 2) 8CH 2NH 2Make 1.35g (94%) title compound, be colorless solid.ESI-MS(m/z)：315[M+H] +。
Embodiment 3Boc-Gly-NHCH 2(CH 2) 10CH 3Preparation
According to the method for embodiment 1 by 0.80g (4.57mmol) Boc-Gly and 0.85g (4.57mmol) CH 3(CH 2) 10CH 2NH 2Make 1.47g (94%) title compound, be colorless solid.ESI-MS(m/z)：343[M+H] +。
Embodiment 4Boc-Gly-NHCH 2(CH 2) 12CH 3Preparation
According to the method for embodiment 1 by 0.80g (4.57mmol) Boc-Gly and 0.98g (4.57mmol) CH 3(CH 2) 12CH 2NH 2Make 1.61g (95%) title compound, be colorless solid.ESI-MS(m/z)：371[M+H] +。
Embodiment 5Boc-Gly-NHCH 2(CH 2) 14CH 3Preparation
According to the method for embodiment 1 by 0.80g (4.57mmol) Boc-Gly and 1.10g (4.57mmol) CH 3(CH 2) 14CH 2NH 2Make 1.76g (97%) title compound, be colorless solid.ESI-MS(m/z)：399[M+H] +。
Embodiment 6Boc-Gly-NHCH 2(CH 2) 16CH 3Preparation
According to the method for embodiment 1 by 0.80g (4.57mmol) Boc-Gly and 1.23g (4.57mmol) CH 3(CH 2) 16CH 2NH 2Make 1.75g (90%) title compound, be colorless solid.ESI-MS(m/z)：427[M+H] +。
Embodiment 7HClGly-NHCH 2(CH 2) 6CH 3Preparation
With 1.24g (4.33mmol) Boc-Gly-OCH 2(CH 2) 6CH 3Be dissolved in 80ml 4N hydrogenchloride-ethyl acetate solution, stirring at room 2 hours, TLC (chloroform: methyl alcohol, 5: 1) shows Boc-Gly-OCH 2(CH 2) 6CH 3Disappear, concentrating under reduced pressure is removed ETHYLE ACETATE, and residue adds a small amount of ether repeatedly and carries out concentrating under reduced pressure to remove de-chlorine hydride.Add a small amount of ether at last residue is ground to form 0.92g (95%) title compound, be beige oily matter.ESI-MS(m/z)：187[M+H] +。
Embodiment 8HClGly-NHCH 2(CH 2) 8CH 3Preparation
According to the method for embodiment 7, from 1.35g (4.29mmol) Boc-Gly-NHCH 2(CH 2) 8CH 3Make 1.03g (96%) title compound, be beige oily matter.ESI-MS(m/z)：215[M+H] +。
Embodiment 9HClGly-NHCH 2(CH 2) 10CH 3Preparation
According to the method for embodiment 7, from 1.47g (4.29mmol) Boc-Gly-NHCH 2(CH 2) 10CH 3Make 1.15g (96%) title compound, be beige oily matter.ESI-MS(m/z)：243[M+H] +。
Embodiment 10HClGly-NHCH 2(CH 2) 12CH 3Preparation
According to the method for embodiment 7, from 1.61g (4.35mmol) Boc-Gly-NHCH 2(CH 2) 12CH 3Make 1.31g (98%) title compound, be the beige solid.ESI-MS(m/z)：271[M+H] +。
Embodiment 11HClGly-NHCH 2(CH 2) 14CH 3Preparation
According to the method for embodiment 7, from 1.76g (4.42mmol) Boc-Gly-NHCH 2(CH 2) 14CH 3Make 1.35g (91%) title compound, be the beige solid.ESI-MS(m/z)：299[M+H] +。
Embodiment 12HClGly-NHCH 2(CH 2) 16CH 3Preparation
According to the method for embodiment 7, from 1.75g (4.11mmol) Boc-Gly-NHCH 2(CH 2) 16CH 3Make 1.40g (94%) title compound, be the beige solid.ESI-MS(m/z)：327[M+H] +。
Embodiment 13Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 6CH 3Preparation
According to the method for embodiment 1 by 1.33g (4.13mmol) Boc-Asp (OBzl) and 0.92g (4.13mmol) HClGly-NHCH 2(CH 2) 6CH 3Make 1.93g (95%) title compound, be faint yellow oily thing.ESI-MS(m/z)：492[M+H] +，[α] 20 D＝-15.2(c＝1.0，CH 3OH)。
Embodiment 14Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 8CH 3Preparation
According to the method for embodiment 1 by 1.33g (4.11mmol) Boc-Asp (OBzl) and 1.03g (4.11mmol) HClGly-NHCH 2(CH 2) 8CH 3Make 1.94g (91%) title compound, be faint yellow oily thing.ESI-MS(m/z)：520[M+H] +，[α] 20 D＝-16.2(c＝1.0，CH 3OH)。
Embodiment 15Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 10CH 3Preparation
According to the method for embodiment 1 by 1.33g (4.13mmol) Boc-Asp (OBzl) and 1.15g (4.13mmol) HClGly-NHCH 2(CH 2) 10CH 3Make 2.17g (96%) title compound, be colorless oil.ESI-MS(m/z)：548[M+H] +，[α] 20 D＝-15.8(c＝1.0，CH 3OH)。
Embodiment 16Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 12CH 3Preparation
According to the method for embodiment 1 by 1.38g (4.27mmol) Boc-Asp (OBzl) and 1.31g (4.27mmol) HClGly-NHCH 2(CH 2) 12CH 3Make 2.09g (85%) title compound, be faint yellow oily thing.ESI-MS(m/z)：576[M+H] +，[α] 20 D＝-14.1(c＝1.0，CH 3OH)。
Embodiment 17Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 14CH 3Preparation
According to the method for embodiment 1 by 1.31g (4.04mmol) Boc-Asp (OBzl) and 1.35g (4.04mmol) HClGly-HNCH 2(CH 2) 14CH 3Make 2.17g (89%) title compound, be faint yellow solid.ESI-MS(m/z)：604[M+H] +，[α] 20 D＝-5.4(c＝1.0，CH 3OH)。
Embodiment 18Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 16CH 3Preparation
According to the method for embodiment 1 by 1.25g (3.86mmol) Boc-Asp (OBzl) and 1.40g (3.86mmol) HClGly-NHCH 2(CH 2) 16CH 3Make 2.09g (86%) title compound, be faint yellow solid.ESI-MS(m/z)：632[M+H] +，[α] 20 D＝-1.3(c＝1.0，CH 3OH)。
Embodiment 19HClAsp (OBzl)-Gly-NHCH 2(CH 2) 6CH 3Preparation
According to the method for embodiment 7, from 1.93g (3.93mmol) Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 6CH 3Make 1.68g (91%) title compound, be the beige solid.ESI-MS(m/z)：392[M+H] +，[α] 20 D＝4.0(c＝1.0，CH 3OH)。
Embodiment 20HClAsp (OBzl)-Gly-NHCH 2(CH 2) 8CH 3Preparation
According to the method for embodiment 7, from 1.94g (3.74mmol) Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 8CH 3Make 1.64g (96%) title compound, be the beige solid.ESI-MS(m/z)：420[M+H] +，[α] 20 D＝2.8(c＝1.0，CH 3OH)。
Embodiment 21HClAsp (OBzl)-Gly-NHCH 2(CH 2) 10CH 3Preparation
According to the method for embodiment 7, from 2.17g (3.97mmol) Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 10CH 3Make 1.77g (92%) title compound, be the beige solid.ESI-MS(m/z)：448[M+H] +，[α] 20 D＝15.8(c＝1.0，CH 3OH)。
Embodiment 22HClAsp (OBzl)-Gly-NHCH 2(CH 2) 12CH 3Preparation
According to the method for embodiment 7, from 2.09g (3.63mmol) Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 12CH 3Make 1.76g (94%) title compound, be the beige solid.ESI-MS(m/z)：476[M+H] +，[α] 20 D＝2.8(c＝1.0，CH 3OH)。
Embodiment 23HClAsp (OBzl)-Gly-NHCH 2(CH 2) 14CH 3Preparation
According to the method for embodiment 7, from 2.17g (3.60mmol) Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 14CH 3Make 185g (95%) title compound, be the beige solid.ESI-MS(m/z)：504[M+H] +，[α] 20 D＝3.0(c＝1.0，CH 3OH)。
Embodiment 24HClAsp (OBzl)-Gly-NHCH 2(CH 2) 16CH 3Preparation
According to the method for embodiment 7, from 2.09g (3.31mmol) Boc-Asp (OBzl)-Gly-NHCH 2(CH 2) 16CH 3Make 1.82g (97%) title compound, be the beige solid.ESI-MS(m/z)：532[M+H] +，[α] 20 D＝2.4(c＝1.0，CH 3OH)。
Embodiment 25Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 6CH 3Preparation
According to the method for embodiment 1 by 1.32g (3.93mmol) Boc-Glu (OBzl) and 1.68g (3.93mmol) HClAsp (OBzl)-Gly-NHCH 2(CH 2) 6CH 3Make, the gained compound gets water white transparency oily thing 1.90g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=100: 1, yield are 68%.ESI-MS(m/z)：711[M+H] +，[α] 20 D＝-19.1(c＝1.0，CH 3OH)。
Embodiment 26Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 8CH 3Preparation
According to the method for embodiment 1 by 1.21g (3.60mmol) Boc-Glu (OBzl) and 1.64g (3.60mmol) HClAsp (OBzl)-Gly-NHCH 2(CH 2) 8CH 3Make, the gained compound gets water white transparency oily thing 1.81g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=100: 1, yield are 68%.ESI-MS(m/z)：739[M+H] +，[α] 20 D＝-36.4(c＝1.0，CH 3OH)。
Embodiment 27Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 10CH 3Preparation
According to the method for embodiment 1 by 1.23g (3.66mmol) Boc-Glu (OBzl) and 1.77g (3.66mmol) HClAsp (OBzl)-Gly-NHCH 2(CH 2) 10CH 3Make, the gained compound gets water white transparency oily thing 1.71g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=100: 1, yield are 61%.ESI-MS(m/z)：767[M+H] +，[α] 20 D＝-24.6(c＝1.0，CH 3OH)。
Embodiment 28Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 12CH 3Preparation
According to the method for embodiment 1 by 1.16g (3.44mmol) Boc-Glu (OBzl) and 1.76g (3.44mmol) HClAsp (OBzl)-Gly-NHCH 2(CH 2) 12CH 3Make, the gained compound gets water white transparency oily thing 1.78g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=100: 1, yield are 65%.ESI-MS(m/z)：795[M+H] +，[α] 20 D＝-31.8(c＝1.0，CH 3OH)。
Embodiment 29Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 14CH 3Preparation
According to the method for embodiment 1 by 1.16g (3.43mmol) Boc-Glu (OBzl) and 1.85g (3.43mmol) HClAsp (OBzl)-Gly-NHCH 2(CH 2) 14CH 3Make, the gained compound gets water white transparency oily thing 2.00g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=100: 1, yield are 71%.ESI-MS(m/z)：823[M+H] +，[α] 20 D＝-13.4(c＝1.0，CH 3OH)。
Embodiment 30Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 16CH 3Preparation
According to the method for embodiment 1 by 1.08g (3.21mmol) Boc-Glu (OBzl) and 1.82g (3.21mmol) HClAsp (OBzl)-Gly-NHCH 2(CH 2) 16CH 3Make, the gained compound gets water white transparency oily thing 1.88g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=100: 1, yield are 69%.ESI-MS(m/z)：851[M+H] +，[α] 20 D＝-3.0(c＝1.0，CH 3OH)。
Embodiment 31HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 6CH 3Preparation
According to the method for embodiment 7, from 1.51g (2.13mmol) Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 6CH 3Make 1.28g (93%) title compound, be colorless solid.ESI-MS(m/z)：611[M+H] +。
Embodiment 32HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 8CH 3Preparation
According to the method for embodiment 7, from 1.41mg (1.91mmol) Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 8CH 3Make 1.16g (90%) title compound, be colorless solid.ESI-MS(m/z)：641[M+H] +。
Embodiment 33HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 10CH 3Preparation
According to the method for embodiment 7, from 1.60mg (2.09mmol) Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 10CH 3Make 1.36g (93%) title compound, be colorless solid.ESI-MS(m/z)：669[M+H] +。
Embodiment 34HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 12CH 3Preparation
According to the method for embodiment 7, from 1.52mg (1.91mmol) Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 12CH 3Make 1.20g (86%) title compound, be colorless solid.ESI-MS(m/z)：697[M+H] +。
Embodiment 35HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 14CH 3Preparation
According to the method for embodiment 7, from 1.65g (2.01mmol) Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 14CH 3Make 1.37g (90%) title compound, be colorless solid.ESI-MS(m/z)：725[M+H] +。
Embodiment 36HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 16CH 3Preparation
According to the method for embodiment 7, from 1.56g (1.84mmol) Boc-Glu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 16CH 3Make 1.27g (88%) title compound, be colorless solid.ESI-MS(m/z)：751[M+H] +。
Embodiment 37Glu-Asp-Gly-NHCH 2(CH 2) 6CH 3Preparation (7a)
With 1.28g (1.98mmol) HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 6CH 3With dissolve with ethanol, add an amount of Pd/C (about 5%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried.Make 751mg (82%) title compound, be colorless solid.ESI-MS(m/z)：429[M-H] -，[α] 20 D＝-9.6(c＝1.0，CH 3OH)，M.p.：104.7-106.4℃。
Embodiment 38Glu-Asp-Gly-NHCH 2(CH 2) 8CH 3Preparation (7b)
With 1.08g (1.60mmol) HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 8CH 3With dissolve with ethanol, add an amount of Pd/C (about 5%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried.Make 699mg (80%) title compound, be colorless solid.ESI-MS(m/z)：457[M-H] -，[α] 20 D＝-6.1(c＝1.0，CH 3OH)，M.p.：89.7-90.1℃。
Embodiment 39Glu-Asp-Gly-NHCH 2(CH 2) 10CH 3Preparation (7c)
With 1.00mg (1.44mmol) HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 10CH 3With dissolve with ethanol, add an amount of Pd/C (about 5%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried.Make 853mg (84%) title compound, be colorless solid.ESI-MS(m/z)：485[M-H] -，[α] 20 D＝-5.5(c＝1.0，CH 3OH)，M.p.：87.4-89.1℃。
Embodiment 40Glu-Asp-Gly-NHCH 2(CH 2) 12CH 3Preparation (7d)
With 1.30g (1.78mmol) HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 12CH 3With dissolve with ethanol, add an amount of Pd/C (about 5%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried.Make 844mg (86%) title compound, be colorless solid.ESI-MS(m/z)：513[M-H] -，[α] 20 D＝-2.8(c＝1.0，CH 3OH)，M.p.：84.7-85.7℃。
Embodiment 41Glu-Asp-Gly-NHCH 2(CH 2) 14CH 3Preparation (7e)
With 1.26g (1.66mmol) HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 14CH 3With dissolve with ethanol, add an amount of Pd/C (about 5%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried.Make 882mg (81%) title compound, be colorless solid.ESI-MS(m/z)：541[M-H] -，[α] 20 D＝-2.2(c＝1.0，CH 3OH)，M.p.：78.3-79.5℃。
Embodiment 42Glu-Asp-Gly-NHCH 2(CH 2) 16CH 3Preparation (7f)
With 1.17g (1.48mmol) HClGlu (OBzl)-Asp (OBzl)-Gly-NHCH 2(CH 2) 16CH 3With dissolve with ethanol, add an amount of Pd/C (about 5%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried.Make 828mg (79%) title compound, be colorless solid.ESI-MS(m/z)：569[M-H] -，[α] 20 D＝-2.9(c＝1.0，CH 3OH)，M.p.：79.4-80.8℃。
Test Example 1 saturated fatty streptamine Glu-Asp-Gly tripeptide amide 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, uses 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 SPL, (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(tric)fraction 2Cultivate 4h for 37 ℃ in the incubator of saturated humidity.Add different concns saturated fatty streptamine Glu-Asp-Gly tripeptide amide (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.Cultivate behind the 48h with the restraining effect of mtt assay detection compound SPL.
Calculate the restraining effect of different concns saturated fatty streptamine Glu-Asp-Gly tripeptide amide 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 streptamine Glu-Asp-Gly tripeptide amide of the present invention has clear and definite restraining effect to mice spleen lymphocytes proliferation.
Table 1 saturated fatty streptamine Glu-Asp-Gly tripeptide amide is to the effect of mouse spleen lymphocyte mitogen inhibition of proliferation
Figure GSB00000669357900111
Test Example 2 saturated fatty streptamine Glu-Asp-Gly tripeptide amides are to the restraining effect of macrophage phagocytic
Growth conditions is good, as to be in logarithmic phase Ana-1 mouse macrophage is 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, add through the saturated fatty streptamine Glu-Asp-Gly of sterilising treatment tripeptide amide (1 * 10 by preset concentration gradient -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, 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 with 2-3 time (removing not by the toluylene red of macrophage phagocytic) of PBS buffered soln washing, (ethanol: 100 μ l acetate=1: 1), 4 ℃ are spent the night, ELIASA 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 streptamine Glu-Asp-Gly tripeptide amide 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 streptamine Glu-Asp-Gly tripeptide amide is engulfed the Ana-1 mouse macrophage
Test Example 3 saturated fatty streptamine Glu-Asp-Gly tripeptide amides are in the self-assembly of nanometer level
Saturated fatty streptamine Glu-Asp-Gly tripeptide amide 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 ability self-assembly granulating is through being stabilized in the nanometer ball of 38.9-1030nm in the saturated fatty streptamine Glu-Asp-Gly tripeptide amide aqueous solution of the present invention.
The nanometer ball particle diameter of table 3 saturated fatty streptamine Glu-Asp-Gly tripeptide amide self-assembly in water
Figure GSB00000669357900131

Claims (5)

1. 6 of general formula I kinds of saturated fatty streptamine Glu-Asp-Gly tripeptide amides with immunosuppressive activity:
Glu-Asp-Gly-NH-CH 2-(CH 2)nCH 3 I
N=6 in the general formula I, 8,10,12,14 or 16.
2. method for preparing the fatty streptamine Glu-Asp-Gly tripeptide amide of claim 1 comprises:
(1) with the glycocoll and the condensation of saturated fatty amine of the protection of N end protection base, said saturated fatty amine is CH 3(CH 2) nCH 2NH 2, n=6,8,10,12,14 or 16;
(2) slough N end protection base and obtain saturated fatty amine glycocoll acid amides;
(3), the protection midbody of saturated fatty amine glycocoll acid amides and aspartic acid, the protection midbody of L-glutamic acid are progressively connect saturated fatty amine three peptide conjugates of the synthetic protection base of peptide protection according to existing liquid phase synthetic technology;
(4) the N end of sloughing saturated fatty amine three peptide conjugates of protection base protection successively protects base and C end protection base to obtain target compound.
3. 6 of claim 1 kinds of saturated fatty streptamine Glu-Asp-Gly tripeptide amide application in preparation immunosuppressor class medicine.
4. 6 of claim 1 kinds of saturated fatty streptamine Glu-Asp-Gly tripeptide amides are as the purposes of the formulation materials of preparation micro emulsion or lipidosome drug carrier.
5. 6 of claim 1 kinds of saturated fatty streptamine Glu-Asp-Gly tripeptide amides are as the purposes in the targeting preparation material of preparation micro emulsion, lipidosome drug carrier.
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