CN101899088A - Saturated fat chain acid His-Gly-AA tripeptide amide, and synthesis method and use thereof - Google Patents

Saturated fat chain acid His-Gly-AA tripeptide amide, and synthesis method and use thereof Download PDF

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CN101899088A
CN101899088A CN2009100853200A CN200910085320A CN101899088A CN 101899088 A CN101899088 A CN 101899088A CN 2009100853200 A CN2009100853200 A CN 2009100853200A CN 200910085320 A CN200910085320 A CN 200910085320A CN 101899088 A CN101899088 A CN 101899088A
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obzl
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赵明
彭师奇
赵淑锐
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Capital Medical University
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Capital Medical University
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Abstract

The invention relates to 12 saturated fat chain acid His-Gly-AA tripeptide amide conjugates having immunosuppressive activity in a general formula I: CH3(CH2)n-CO-His-Gly-AA, wherein AA is Glu or Lys; and n in the saturated fat chain acid is equal to 6, 8, 10, 12, 14 or 16. The invention relates to a preparation method for the tripeptide amide conjugates and use of the tripeptide amide conjugates as immunosuppressive agents. Experimental results that the saturated fat chain acid His-Gly-AA tripeptide amide has the effects of inhibiting splenic lymphocyte cell mitogen breeder reaction and inhibiting macrophage phagocytosis activities show that the compounds have good immunodepression effect and can be clinically used as the immunosuppressive agents.

Description

Saturated fat chain acid His-Gly-AA tripeptide amide and synthetic method thereof and application
Technical field
The present invention relates to the saturated fat chain acid tripeptide amide, relate in particular to a series of saturated fat chain acid His-Gly-AA tripeptide amides with immunosuppressive activity, also relate to the preparation method of these saturated fat chain acid tripeptide amides 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 acid amides that generation is puted together in help a small child urinate by holding his legs apart toxin peptide and aliphatic chain acid 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 aliphatic chain acid are puted together, and obtains having the saturated fat chain acid tripeptide amide conjugate of immunosuppressive activity.
One of purpose of the present invention is achieved through the following technical solutions:
12 kinds of saturated fat chain acid His-Gly-AA tripeptide amides of general formula I with immunosuppressive activity
CH 3(CH 2) n-CO-His-Gly-AA 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 fat chain acid His-Gly-AA tripeptide amide 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 fat chain acid His-Gly-AA tripeptide amide of immunosuppressive activity, this method comprises:
(1) according to existing liquid phase synthetic technology, by progressively connecing the protection intermediate of peptide synthetic His-Gly-AA, wherein AA is L-glutamic acid or Methionin;
(2) N that sloughs the protection intermediate of His-Gly-AA holds protecting group;
(3) the His-Gly-AA intermediate and the saturated fatty acid condensation of C being held protecting group protect, described saturated fatty acid is CH 3(CH 2) nCOOH, n=6,8,10,12,14 or 16;
(4) slough C end protecting group and 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, condensation, deprotection is the conventional and technique known of this area.
When AA was Glu, the preparation method of described saturated fatty acid 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 γ-benzyl ester-benzyl glutamate and the condensation of N-t-butoxycarbonyl glycine, generate N-tertbutyloxycarbonyl glycyl γ-benzyl ester-benzyl glutamate;
(4) in hydrogenchloride-ethyl acetate, N-tertbutyloxycarbonyl glycyl γ-benzyl ester-benzyl glutamate is removed the tertiary butyloxycarbonyl protecting group, generate glycyl γ-benzyl ester-benzyl glutamate;
(5) in anhydrous THF, in the presence of DCC and HOBt with glycyl γ-benzyl ester-benzyl glutamate and N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl-Histidine condensation generates N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl-histidyl-glycyl γ-benzyl ester-benzyl glutamate;
(6) in the presence of hydrogenchloride-ethyl acetate with N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl-histidyl-glycyl γ-benzyl ester-benzyl glutamate removes tertbutyloxycarbonyl and generates histidyl-glycyl γ-benzyl ester-benzyl glutamate;
(7) in anhydrous THF, in the presence of DCC and HOBt,, generate saturated fatty acid acyl histidyl-glycyl γ-benzyl ester-benzyl glutamate with histidyl-glycyl γ-benzyl ester-benzyl glutamate and saturated fatty acid condensation;
(8) in dehydrated alcohol, in the presence of Pd/C,, generate saturated fatty acid acyl histidyl-glycyl L-glutamic acid with histidyl-glycyl γ-benzyl ester-benzyl 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 ε-carbobenzoxy-(Cbz) Methionin benzyl ester and the condensation of N-t-butoxycarbonyl glycine generate N-tertbutyloxycarbonyl glycyl N ε-carbobenzoxy-(Cbz) Methionin benzyl ester;
(4) in hydrogenchloride-ethyl acetate with N-tertbutyloxycarbonyl glycyl N ε-carbobenzoxy-(Cbz) Methionin benzyl ester removes the tertiary butyloxycarbonyl protecting group, generates glycyl N ε-carbobenzoxy-(Cbz) Methionin benzyl ester;
(5) in anhydrous THF, in the presence of DCC and HOBt with glycyl N ε-carbobenzoxy-(Cbz) Methionin benzyl ester and N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl-Histidine condensation generates N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl-histidyl-glycyl N ε-carbobenzoxy-(Cbz) Methionin benzyl ester;
(6) in the presence of hydrogenchloride-ethyl acetate with N-tertbutyloxycarbonyl-N Im-tertbutyloxycarbonyl-histidyl-glycyl N ε-carbobenzoxy-(Cbz) Methionin benzyl ester removes tertbutyloxycarbonyl, generates histidyl-glycyl N ε-carbobenzoxy-(Cbz) Methionin benzyl ester;
(7) in anhydrous THF, in the presence of DCC and HOBt with histidyl-glycyl N ε-carbobenzoxy-(Cbz) Methionin benzyl ester and saturated fatty acid condensation generate saturated fatty acid acyl histidyl-glycyl N ε-carbobenzoxy-(Cbz) Methionin benzyl ester;
(8) in dehydrated alcohol, in the presence of Pd/C with fatty acid acyl histidyl-glycyl N ε-carbobenzoxy-(Cbz) Methionin benzyl ester hydrogenolysis generates saturated fatty acid acyl histidyl-glycyl Methionin.
Experimental result shows that 12 kinds of saturated fat chain acid His-Gly-AA tripeptide amides 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 113-4720nm in the saturated fat chain acid His-Gly-AA tripeptide amide 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 fat chain acid His-Gly-Glu tripeptide amide conjugate 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 26a n=6,6b n=8,6c n=10,6d n=12,6e n=14,6f n=16.
Fig. 2 is 6 kinds of synthetic routes with saturated fat chain acid His-Gly-Lys tripeptide amide conjugate 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 212a n=6,12b n=8,12c n=10,12d n=12,12e n=14,12f 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-Gly-Glu (OBzl)-OBzl
3.50g (20.00mmol) Boc-Gly is dissolved in the anhydrous THF of 200ml, in the solution that obtains, adds 2.72g (20.00mmol) N-hydroxy benzo triazole (HOBt) under the ice bath, and it is dissolved fully.Add 4.96g (24.00mmol) dicyclohexylcarbodiimide (DCC) after 10 minutes.Obtain reaction solution (I), stand-by.The following 10.38g of ice bath (20.00mmol) TosHGlu (OBzl)-OBzl 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), stand-by.The following reaction solution of ice bath (I) adds in the reaction solution (II), stirs 1h under the condition of ice bath, stirring at room 12h again, and TLC (chloroform/methanol, 10: 1) shows that Boc-Gly-OH 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 8.91g (92%) title compound, are beige oily matter.ESI-MS(m/z)：485[M+H] +。
Embodiment 2 preparation HClGly-Glu (OBzl)-OBzl
8.91g (18.38mmol) Boc-Gly-Glu (OBzl)-OBzl is dissolved in 200ml 4mol/l hydrogenchloride-ethyl acetate solution, stirring at room 2 hours, TLC (chloroform: methyl alcohol, 5: 1) show that raw material point disappears, 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 hydrogen chloride gas.Add a small amount of ether at last residue is ground to form 6.94g (90%) title compound, be the colorless solid powder, be directly used in next step reaction.ESI-MS(m/z)：385[M+H] +。
Embodiment 3 preparation Boc-His (Boc)-Gly-Glu (OBzl)-OBzl
Method according to embodiment 1 gets beige oily matter by 5.42g (7.63mmol) Boc-His (Boc) and 6.94g (15.26mmol) HClGly-Glu (OBzl)-OBzl.The gained compound gets colorless oil target 8.24g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=100: 1, yield are 75%.ESI-MS(m/z)：722[M+H] +，[α] 20 D＝-25.2(c＝1.0，CH 3OH)。
Embodiment 4 preparation HClHis-Gly-Glu (OBzl)-OBzl
Method 8.24g (11.42mmol) Boc-His (Boc)-Gly-Glu (OBzl)-OBzl according to embodiment 2 makes 6.05g (95%) title compound, is the beige solid.ESI-MS(m/z)：522[M+H] +。
Embodiment 5 preparation CH 3(CH 2) 6CO-His-Gly-Glu (OBzl)-OBzl
According to the method for embodiment 1 by 0.26g (1.79mmol) CH 3(CH 2) 6CO 2H and 1.00g (1.79mmol) HClHis-Gly-Glu (OBzl)-OBzl makes title compound, is yellow solid.The gained compound gets colorless solid powder 846mg through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 73%.ESI-MS(m/z)：648[M+H] +，[α] 20 D＝-7.2(c＝1.0，CH 3OH)，M.p.：65.3-65.8℃。
Embodiment 6 preparation CH 3(CH 2) 8CO-His-Gly-Glu (OBzl)-OBzl
According to the method for embodiment 1 by 0.31g (1.79mmol) CH 3(CH 2) 8CO 2H and 1.00g (1.79mmol) HClHis-Gly-Glu (OBzl)-OBzl makes title compound, is yellow solid.The gained compound gets colorless solid powder 810mg through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 67%.ESI-MS(m/z)：676[M+H] +，[α] 20 D＝-4.5(c＝1.0，CH 3OH)，M.p.：67.2-68.9℃。
Embodiment 7 preparation CH 3(CH 2) 10CO-His-Gly-Glu (OBzl)-OBzl
According to the method for embodiment 1 by 0.36g (1.79mmol) CH 3(CH 2) 10CO 2H and 1.00g (1.79mmol) HClHis-Gly-Glu (OBzl)-OBzl makes title compound, is faint yellow solid.The gained compound gets colorless solid powder 868mg through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 69%.ESI-MS(m/z)：704[M+H] +，[α] 20 D＝-9.7(c＝1.0，CH 3OH)，M.p.：68.9-70.4℃。
Embodiment 8 preparation CH 3(CH 2) 12CO-His-Gly-Glu (OBzl)-OBzl
According to the method for embodiment 1 by 0.41g (1.79mmol) CH 3(CH 2) 12CO 2H and 1.00g (1.79mmol) HClHis-Gly-Glu (OBzl)-OBzl makes title compound, is yellow solid.The gained compound gets colorless solid powder 955mg through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 73%.ESI-MS(m/z)：732[M+H] +，[α] 20 D＝-3.2(c＝1.0，CH 3OH)，M.p.：68.9-69.9℃。
Embodiment 9 preparation CH 3(CH 2) 14CO-His-Gly-Glu (OBzl)-OBzl
According to the method for embodiment 1 by 0.44g (1.71mmol) CH 3(CH 2) 14CO 2H and 1.00g (1.71mmol) HClHis-Gly-Glu (OBzl)-OBzl makes title compound, is yellow solid.The gained compound gets colorless solid powder 802mg through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 59%.ESI-MS(m/z)：760[M+H] +，[α] 20 D＝-12.5(c＝1.0，CH 3OH)，M.p.：71.5-72.9℃。
Embodiment 10 preparation CH 3(CH 2) 16CO-His-Gly-Glu (OBzl)-OBzl
According to the method for embodiment 1 by 0.51g (1.79mmol) CH 3(CH 2) 16CO 2H and 1.00g (1.79mmol) HClHis-Gly-Glu (OBzl)-OBzl makes title compound, is yellow solid.The gained compound gets colorless solid powder 874mg through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 62%.ESI-MS(m/z)：788[M+H] +，[α] 20 D＝-20.9(c＝1.0，CH 3OH)，M.p.：71.5-73.4℃。
Embodiment 11 preparation CH 3(CH 2) 6CO-His-Gly-Glu (6a)
With 700mg (1.08mmol) CH 3(CH 2) 6CO-His-Gly-Glu (OBzl)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 35mgPd/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, and residue grinds with sherwood oil repeatedly, makes 444mg (88%) title compound, is colorless solid.ESI-MS(m/z)：465[M-H] -，[α] 20 D＝-3.0(c＝1.0，CH 3OH)，M.p.：126.0-127.8℃。
Embodiment 12 preparation CH 3(CH 2) 8CO-His-Gly-Glu (6b)
With 800mg (1.19mmol) CH 3(CH 2) 8CO-His-Gly-Glu (OBzl)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 40mgPd/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, and residue grinds with sherwood oil repeatedly, makes 462mg (79%) title compound, is colorless solid.ESI-MS(m/z)：493[M-H] -；[α] 20 D＝-8.1(c1，CH 3OH)；M.p.：119.5-120.5℃。
Embodiment 13 preparation CH 3(CH 2) 10CO-His-Gly-Glu (6c)
With 800mg (1.14mmol) CH 3(CH 2) 10CO-His-Gly-Glu (OBzl)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 40mgPd/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, and residue grinds with sherwood oil repeatedly, makes 456mg (77%) title compound, is colorless solid.ESI-MS(m/z)：521[M-H] -，[α] 20 D＝-18.0(c＝1.0，CH 3OH)，M.p.：123.6-125.2℃。
Embodiment 14 preparation CH 3(CH 2) 12CO-His-Gly-Glu (6d)
With 900mg (1.23mmol) CH 3(CH 2) 12CO-His-Gly-Glu (OBzl)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 45mgPd/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, and residue grinds with sherwood oil repeatedly, makes 472mg (70%) title compound, is colorless solid.ESI-MS(m/z)：549[M-H] -，[α] 20 D＝-17.7(c＝1.0，CH 3OH)，M.p.：131.2-132.3℃。
Embodiment 15 preparation CH 3(CH 2) 14CO-His-Gly-Glu (6e)
With 800mg (1.05mmol) CH 3(CH 2) 14CO-His-Gly-Glu (OBzl)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 40mgPd/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, and residue grinds with sherwood oil repeatedly, makes 504mg (83%) title compound, is colorless solid.ESI-MS(m/z)：577[M-H] -，[α] 20 D＝-4.2(c＝1.0，CH 3OH)，M.p.：158.1-160.0℃。
Embodiment 16 preparation CH 3(CH 2) 16CO-His-Gly-Glu (6f)
With 900mg (1.14mmol) CH 3(CH 2) 16CO-His-Gly-Glu (OBzl)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 45mgPd/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, and residue grinds with sherwood oil repeatedly, makes 561mg (81%) title compound, is colorless solid.ESI-MS(m/z)：605[M-H] -，[α] 20 D＝-5.7(c＝1.0，CH 3OH)，M.p.：159.7-161.4℃。
Embodiment 17 preparation Boc-Gly-Lys (Z)-OBzl
0.88g (5.0mmol) Boc-Gly is dissolved in the anhydrous THF of 200ml, in the solution that obtains, adds 0.68g (5mmol) N-hydroxy benzo triazole (HOBt) under the ice bath, and it is dissolved fully.Add 1.24g (6mmol) dicyclohexylcarbodiimide (DCC) after 10 minutes.Obtain reaction solution (III), stand-by.The following 2.81g of ice bath (5.0mmol) TosLys (Z)-OBzl (5mmol) 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), stand-by.The following reaction solution of ice bath (III) adds in the reaction solution (IV), stirs 1h under the condition of ice bath, stirring at room 12h again, and TLC (chloroform/methanol, 10: 1) shows that Boc-Gly 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 2.32g (88%) title compound, are the beige solid.ESI-MS(m/z)：528[M+H] +。
Embodiment 18 preparation HClGly-Lys (Z)-OBzl
2.32g (4.40mmol) Boc-Gly-Lys (Z)-OBzl is dissolved in 100ml 4mol/l hydrogenchloride-ethyl acetate solution, stirring at room 2 hours, TLC (chloroform: methyl alcohol, 5: 1) show that raw material point disappears, 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 hydrogen chloride gas.Add a small amount of ether at last residue is ground to form 1.94g (95%) title compound, be the colorless solid powder, be directly used in next step reaction.ESI-MS(m/z)：428[M+H] +。
Embodiment 19 preparation Boc-His (Boc)-Gly-Lys (Z)-OBzl
Method according to embodiment 17 gets beige oily matter by 1.49g (4.18mmol) Boc-His (Boc) and 1.94g (4.18mmol) HClGly-Lys (Z)-OBzl.The gained compound gets colorless oil target 1.87g through purification by silica gel column chromatography, and yield is 58.47%.ESI-MS(m/z)：765[M+H] +，[α] 20 D＝27.4(c＝1.0，CH 3OH)。
Embodiment 20 preparation HClHis-Gly-Lys (Z)-OBzl
Method according to embodiment 18 makes 1.41g (95%) title compound by 1.87g (2.45mmol) Boc-His (Boc)-Gly-Lys (Z)-OBzl, is the beige solid.ESI-MS(m/z)：565[M+H] +。
Embodiment 21 preparation CH 3(CH 2) 6CO-His-Gly-Lys (Z)-OBzl
According to the method for embodiment 17 by 0.41g (2.82mmol) CH 3(CH 2) 6CO 2H and 1.41g (2.35mmol) HClHis-Gly-Lys (Z)-OBzl makes title compound, is yellow solid.The gained compound gets colorless solid powder 794mg through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 49%.ESI-MS(m/z)：691[M+H] +，[α] 20 D＝-12.6(c＝1.0，CH 3OH)，M.p.：88.5-89.6℃。
Embodiment 22 preparation CH 3(CH 2) 8CO-His-Gly-Lys (Z)-OBzl
According to the method for embodiment 17 by 0.49g (2.82mmol) CH 3(CH 2) 8CO 2H and 1.41g (2.35mmol) HClHis-Gly-Lys (Z)-OBzl makes title compound, is yellow solid.The gained compound gets colorless solid powder 726mg through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 43%.ESI-MS(m/z)：719[M+H] +，[α] 20 D＝-28.0(c＝1.0，CH 3OH)，M.p.：89.7-90.1℃。
Embodiment 23 preparation CH 3(CH 2) 10CO-His-Gly-Lys (Z)-OBzl
According to the method for embodiment 17 by 0.57g (2.82mmol) CH 3(CH 2) 10CO 2H and 1.41g (2.35mmol) HClHis-Gly-Lys (Z)-OBzl makes title compound, is yellow solid.The gained compound gets colorless solid powder 1.03g through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 59%.ESI-MS(m/z)：747[M+H] +，[α] 20 D＝-14.6(c＝1.0，CH 3OH)，M.p.：89.1-91.0℃。
Embodiment 24 preparation CH 3(CH 2) 12CO-His-Gly-Lys (Z)-OBzl
According to the method for embodiment 17 by 0.65g (2.82mmol) CH 3(CH 2) 12CO 2H and 1.41g (2.35mmol) HClHis-Gly-Lys (Z)-OBzl makes title compound, is yellow solid.The gained compound gets colorless solid powder 854mg through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 47%.ESI-MS(m/z)：775[M+H] +，[α] 20 D＝-20.8(c＝1.0，CH 3OH)，M.p.：88.6-90.4℃。
Embodiment 25 preparation CH 3(CH 2) 14CO-His-Gly-Lys (Z)-OBzl
According to the method for embodiment 17 by 0.72g (2.82mmol) CH 3(CH 2) 14CO 2H and 1.41g (2.35mmol) HClHis-Gly-Lys (Z)-OBzl makes title compound, is yellow solid.The gained compound gets colorless solid powder 998mg through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 53%.ESI-MS(m/z)：803[M+H] +，[α] 20 D＝-23.4(c＝1.0，CH 3OH)，M.p.：86.4-87.1℃。
Embodiment 26 preparation CH 3(CH 2) 16CO-His-Gly-Lys (Z)-OBzl
According to the method for embodiment 17 by 0.80g (2.82mmol) CH 3(CH 2) 16CO 2H and 1.41g (2.35mmol) HClHis-Gly-Lys (Z)-OBzl makes title compound, is yellow solid.The gained compound gets colorless solid powder 858mg through purification by silica gel column chromatography, purification condition: chloroform: methyl alcohol=50: 1, yield are 44%.ESI-MS(m/z)：831[M+H] +，[α] 20 D＝-22.3(c＝1.0，CH 3OH)，M.p.：84.5-86.5℃。
Embodiment 27 preparation CH 3(CH 2) 6CO-His-Gly-Lys (12a)
With 691mg (1mmol) CH 3(CH 2) 6CO-His-Gly-Lys (Z)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 35mg 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, and residue grinds with sherwood oil repeatedly, makes 376mg (79%) title compound, is colorless solid.ESI-MS(m/z)：464[M-H] -，[α] 20 D＝-3.5(c＝1.0，CH 3OH)，M.p.：127.1-128.7℃。
Embodiment 28 preparation CH 3(CH 2) 8CO-His-Gly-Lys (12b)
With 526mg (0.73mmol) CH 3(CH 2) 8CO-His-Gly-Lys (Z)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 30mg 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, and residue grinds with sherwood oil repeatedly, makes 263mg (73%) title compound, is colorless solid.ESI-MS(m/z)：492[M-H] -；[α] 20 D＝-4.6(c1，CH 3OH)；M.p.：132.3-133.5℃。
Embodiment 29 preparation CH 3(CH 2) 10CO-His-Gly-Lys (12c)
With 747mg (1mmol) CH 3(CH 2) 10CO-His-Gly-Lys (Z)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 40mg 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, and residue grinds with sherwood oil repeatedly, makes 400mg (77%) title compound, is colorless solid.ESI-MS(m/z)：520[M-H] -，[α] 20 D＝-8.3(c＝1.0，CH 3OH)，M.p.：133.8-135.3℃。
Embodiment 30 preparation CH 3(CH 2) 12CO-His-Gly-Lys (12d)
With 654mg (0.84mmol) CH 3(CH 2) 12CO-His-Gly-Lys (Z)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 35mg 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, and residue grinds with sherwood oil repeatedly, makes 361mg (78%) title compound, is colorless solid.ESI-MS(m/z)：548[M-H] -，[α] 20 D＝-37.1(c＝1.0，CH 3OH)，M.p.：146.0-147.2℃。
Embodiment 31 preparation CH 3(CH 2) 14CO-His-Gly-Lys (12e)
With 700mg (0.87mmol) CH 3(CH 2) 14CO-His-Gly-Lys (Z)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 35mg 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, and residue grinds with sherwood oil repeatedly, makes 406mg (81%) title compound, is colorless solid.ESI-MS(m/z)：576[M-H] -，[α] 20 D＝-8.4(c＝1.0，CH 3OH)，M.p.：134.5-135.1℃。
Embodiment 32 preparation CH 3(CH 2) 16CO-His-Gly-Lys (12f)
With 688mg (0.83mmol) CH 3(CH 2) 16CO-His-Gly-Lys (Z)-OBzl places the 100ml eggplant-shape bottle, with dissolve with ethanol, add 35mg 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, and residue grinds with sherwood oil repeatedly, makes 350mg (70%) title compound, is colorless solid.ESI-MS(m/z)：604[M-H] -，[α] 20 D＝-3.6(c＝1.0，CH 3OH)，M.p.：109.1-110.9℃。
Test example 1 saturated fat chain acid His-Gly-AA 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, 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 fat chain acid His-Gly-AA 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.Use the restraining effect of mtt assay detection compound after cultivating 48h to splenic lymphocyte.
Calculate the restraining effect of different concns saturated fat chain acid His-Gly-AA 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 fat chain acid His-Gly-AA tripeptide amide of the present invention has clear and definite restraining effect to mice spleen lymphocytes proliferation.
Table 1 saturated fat chain acid His-Gly-AA tripeptide amide is to the effect of mouse spleen lymphocyte mitogen inhibition of proliferation
Figure B2009100853200D0000111
Test example 2 saturated fat chain acid His-Gly-AA tripeptide amides 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 fat chain acid His-Gly-AA tripeptide amide (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 fat chain acid His-Gly-AA 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 fat chain acid His-Gly-AA tripeptide amide is engulfed the Ana-1 mouse macrophage
Figure B2009100853200D0000121
Test example 3 saturated fat chain acid His-Gly-AA tripeptide amides are in the self-assembly of nanometer level
Saturated fat chain acid His-Gly-AA 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 energy self-assembly granulating is through being stabilized in the nanometer ball of 113-4720nm in the saturated fat chain acid His-Gly-AA tripeptide amide aqueous solution of the present invention.
The nanometer ball particle diameter of table 3 saturated fat chain acid His-Gly-AA tripeptide amide self-assembly in water
Figure B2009100853200D0000131

Claims (5)

1. 12 of general formula I kinds of saturated fat chain acid His-Gly-AA tripeptide amides with immunosuppressive activity:
CH 3(CH 2) n-CO-His-Gly-AA 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 fat chain acid His-Gly-AA tripeptide amide of claim 1 comprises:
(1) according to existing liquid phase synthetic technology, by progressively connecing the protection intermediate of peptide synthetic His-Gly-AA, wherein AA is L-glutamic acid or Methionin;
(2) N that sloughs the protection intermediate of His-Gly-AA holds protecting group;
(3) the His-Gly-AA intermediate and the saturated fatty acid condensation of C being held protecting group protect, described saturated fatty acid is CH 3(CH 2) nCOOH, n=6,8,10,12,14 or 16;
(4) slough C end protecting group and obtain target compound.
3. 12 of claim 1 kinds of saturated fat chain acid His-Gly-AA tripeptide amide application in preparation immunosuppressor class medicine.
4. 12 of claim 1 kinds of saturated fat chain acid His-Gly-AA tripeptide amides are as the purposes of the preparation material of preparation micro emulsion or lipidosome drug carrier.
5. 12 of claim 1 kinds of saturated fat chain acid His-Gly-AA tripeptide amides are as the purposes in the targeting preparation material of preparation micro emulsion, lipidosome drug carrier.
CN2009100853200A 2009-05-26 2009-05-26 Saturated fat chain acid His-Gly-AA tripeptide amide, and synthesis method and use thereof Pending CN101899088A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106083634A (en) * 2016-06-16 2016-11-09 淮阴师范学院 A kind of peptide amphiphile molecule and its preparation method and application
CN112094321A (en) * 2019-06-18 2020-12-18 首都医科大学 His-Gly-Glu modified methotrexate, synthesis, anti-transfer activity and application thereof
CN112898377A (en) * 2019-12-02 2021-06-04 首都医科大学 Dioxane-modified tetrahydrocarboline-3-formyl-The-HGE, preparation thereof, anti-inflammatory activity thereof and application thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106083634A (en) * 2016-06-16 2016-11-09 淮阴师范学院 A kind of peptide amphiphile molecule and its preparation method and application
CN106083634B (en) * 2016-06-16 2017-12-05 淮阴师范学院 A kind of peptide amphiphile molecule and its preparation method and application
CN112094321A (en) * 2019-06-18 2020-12-18 首都医科大学 His-Gly-Glu modified methotrexate, synthesis, anti-transfer activity and application thereof
CN112898377A (en) * 2019-12-02 2021-06-04 首都医科大学 Dioxane-modified tetrahydrocarboline-3-formyl-The-HGE, preparation thereof, anti-inflammatory activity thereof and application thereof
CN112898377B (en) * 2019-12-02 2022-04-22 首都医科大学 Dioxane-modified tetrahydrocarboline-3-formyl-The-HGE, preparation thereof, anti-inflammatory activity thereof and application thereof

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