CN102796170B - Double saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters and preparation method and application thereof - Google Patents

Double saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters and preparation method and application thereof Download PDF

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CN102796170B
CN102796170B CN201110141197.7A CN201110141197A CN102796170B CN 102796170 B CN102796170 B CN 102796170B CN 201110141197 A CN201110141197 A CN 201110141197A CN 102796170 B CN102796170 B CN 102796170B
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tertbutyloxycarbonyl
asp
lys
fatty alcohol
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CN102796170A (en
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彭师奇
赵明
李婕
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Capital Medical University
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Abstract

The invention relates to six saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters which are shown as a general formula I and have immunosuppressive activity, and a preparation method thereof. The general formula I is His-Gly-Lys-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3, wherein n is 6, 8, 10, 12, 14 or 16. By studying inhibition effects of aliphatic chain alcohol on proliferation reaction of a splenic lymphocyte mitogen and the phagocytic activity of a macrophage, and the survival time of a mouse subjected to opisthotic cardiac transplantation, experimental data further shows that the compounds have a good immunosuppressive effect, so the compounds have a wide application prospect in preparation of immunosuppressive medicines.

Description

Two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide ester, its preparation method and application
Technical field
The present invention relates to a kind of compound of synthetic, be particularly related to a kind of 6 kinds of two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters with immunosuppressive activity of general formula I, the preparation method who also relates to them, with and application in preparing immunosuppressive drug, the invention belongs to biomedicine field.
Background technology
Before thousands of year, people just once imagination with normal organ-tissue, replace the organ-tissue of pathology, but transplanting before 20th century is except corneal transplantation, major part is failed.Until the forties in 20th century, the dermatoplastic experimental model of Britain surgeon Mdewaar utilizer rabbit, having proved for the first time the rejection occurring in organ transplantation is a kind of immune response in essence, thereby has established the basis of transplantation immunology.After the eighties, effectively immunosuppressor is as discovery and the application of cyclosporin A and FK506 etc., the survival time of transplant organ is further extended, but because clinical application ubiquity is easily degraded, bioavailability is low, fat-soluble poor, be difficult to see through the shortcomings such as microbial film, so urgently study medicament nanometer packaging material, go to improve these weak points.To be self-assembled into clear and definite nanofibrous structures in widespread attention because of it for peptide amphiphile, from nanotechnology to organizational project, is applied in a lot of fields.And due to the diversity of peptide amphiphile chemical structure be easy to synthesize, will become the study hotspot that biological medicine and technical field of biological material are new.Contriver recognizes, the urotoxin peptide with immunosuppressive activity is the endogenous peptide with immunosuppressive activity of separating in human body.The fat-soluble design philosophy that increases peptide from structural modification is to improve its bioavailability in vivo.According to this conception, contriver finds that urotoxin peptide and aliphatic chain coupling have immunosuppressive activity really.So contriver proposes the present invention.
Summary of the invention
First technical problem to be solved by this invention is that 6 kinds of two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters (representing with 9a-f) with immunosuppressive activity of general formula I are provided;
His-Gly-Lys-Asp[OCH 2(CH 2)nCH 3]-OCH 2(CH 2)nCH 3 I
N=6 in general formula I, 8,10,12,14 or 16;
Wherein, n=6 in 9a; N=8 in 9b; N=10 in 9c; N=12 in 9d; N=14 in 9e; N=16 in 9f;
Second technical problem to be solved by this invention be, 6 kinds of preparation methods with two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters of immunosuppressive activity of general formula I are provided, and concrete comprises the following steps;
(1) at (Boc) 2o and NaOH are converted into N-t-butoxycarbonyl glycine by glycine under existing;
(2) at (Boc) 2o and NaOH are converted into N-tertbutyloxycarbonyl aspartic acid by aspartic acid under existing;
(3) at (Boc) 2o and triethylamine are converted into N-tertbutyloxycarbonyl-N by Histidine under existing im-tertbutyloxycarbonyl-Histidine;
(4) under dicyclohexyl carbonyl diimine (DCC), N-hydroxy benzo triazole (HOBt), anhydrous tetrahydro furan (THF) exist by the saturated fatty alcohol condensation of N-tertbutyloxycarbonyl aspartic acid and doubling dose, generate pair saturated fatty alcohol N-tertbutyloxycarbonyl aspartates;
(5) in hydrogenchloride-ethyl acetate, two saturated fatty alcohol N-tertbutyloxycarbonyl aspartates are removed to tertiary butyloxycarbonyl protecting group, generate two saturated fatty alcohol aspartates;
(4) under DCC, HOBt, anhydrous THF exist by N α-tertbutyloxycarbonyl-N ε-carbobenzoxy-(Cbz) Methionin and two saturated fatty alcohol aspartic acid ester condensations, generate two saturated fatty alcohol N α-tertbutyloxycarbonyl-N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(6) in hydrogenchloride-ethyl acetate by two saturated fatty alcohol N α-tertbutyloxycarbonyl-N ε-carbobenzoxy-(Cbz) lysyl aspartate removes tertiary butyloxycarbonyl protecting group, generates two saturated fatty alcohol N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(7) in anhydrous THF, under DCC and HOBt exist by two saturated fatty alcohol N ε-carbobenzoxy-(Cbz) lysyl aspartate and the condensation of N-t-butoxycarbonyl glycine, generate two saturated fatty alcohol N-tertbutyloxycarbonyl glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(8) under hydrogenchloride-ethyl acetate exists by two saturated fatty alcohol N-tertbutyloxycarbonyl glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate removes tertbutyloxycarbonyl, generates two saturated fatty alcohol glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(9) in anhydrous THF, under DCC and HOBt exist by two saturated fatty alcohol glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate and N-tertbutyloxycarbonyl-N imthe condensation of-tertbutyloxycarbonyl-Histidine, generates two saturated fatty alcohol N-tertbutyloxycarbonyl-N im-tertbutyloxycarbonyl histidyl-glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(10) in hydrogenchloride-ethyl acetate by two saturated fatty alcohol N-tertbutyloxycarbonyl-N im-tertbutyloxycarbonyl histidyl-glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate removes tertbutyloxycarbonyl, generates two saturated fatty alcohol histidyl-glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(11) in dehydrated alcohol, under Pd/C exists by two saturated fatty alcohol histidyl-glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate hydrogenolysis, generates two saturated fatty alcohol histidyl-glycyl lysyl aspartates, obtains.
The 3rd technical problem to be solved by this invention be, evaluate 6 kinds of two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters to mouse spleen lymphocyte the restraining effect to the proliferative response of mitogen;
The 4th technical problem to be solved by this invention is to evaluate the restraining effect of 6 kinds of two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters to macrophage phagocytic activity;
The 5th technical problem to be solved by this invention is to evaluate the autonomous dress performance of 6 kinds of two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters;
The 6th technical problem to be solved by this invention is to evaluate 6 kinds of two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide ester mouse ear rear myocardium tissue's transplanting survival times.
The present invention to the restraining effect of splenic lymphocyte mitogen proliferative response, to the restraining effect of macrophage phagocytic activity and to the survival time after myocardium transplantation after mouse ear, has further shown that compound of the present invention has outstanding immunosuppressive action by the saturated aliphatic chain alcohol described in research.In preparing immune suppressant drug, be with a wide range of applications.
One of purposes of of the present invention pair of saturated aliphatic chain alcohol Glu-Asp-Gly-Asp tetrapeptide ester is the purposes in preparation immunosuppressor;
Two of the purposes of of the present invention pair of saturated aliphatic chain alcohol Glu-Asp-Gly-Asp tetrapeptide ester is the purposes in preparation immunosuppression pharmacome;
Three of the purposes of of the present invention pair of saturated aliphatic chain alcohol Glu-Asp-Gly-Asp tetrapeptide ester is the purposes in preparing micro emulsion or liposome targeted drug.
Accompanying drawing explanation
Fig. 1 is the synthetic route that 6 kinds of general formula I have two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters of immunosuppressive activity;
I) anhydrous THF, DCC, HOBt and NMM; Ii) 4N hydrogenchloride-ethyl acetate solution; Iii) dehydrated alcohol, Pd/C and H 2; 1-9a n=6,1-9b n=8,1-9c n=10,1-9d n=12,1-9e n=14,1-9f n=16;
Fig. 2 is the representative transmission electron microscope photo of the nanometer ball that forms in the aqueous solution of 9d.
Breviary term
THF tetrahydrofuran (THF)
HOBt N-hydroxy benzo triazole
DCC dicyclohexyl carbonyl diimine
DCU dicyclohexylurea (DCU)
NMM N-methylmorpholine
TLC thin-layer chromatography
Boc tertbutyloxycarbonyl
Embodiment
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative completely, and they are only used for the present invention to be specifically described, and not should be understood to limitation of the present invention.
Embodiment 1 preparation Boc-Asp[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3(1a)
1.1g (4.8mmol) Boc-Asp-OH is dissolved in to the anhydrous THF of 20mL, under ice bath, in the solution obtaining, adds 1.5g (1.2mmol) N-hydroxy benzo triazole (HOBt), and it is dissolved completely.After 10 minutes, add 2.3g (1.2mmol) dicyclohexylcarbodiimide (DCC).Obtain reaction solution (I), stand-by.The lower 1.5g of ice bath (1.2mmol) CH 3(CH 2) 6cH 2oH is suspended in the anhydrous THF of 20mL, then adds 1mlN-methylmorpholine (NMM), adjusts pH 8-9.Stir 35 minutes, obtain reaction solution (II), stand-by.The lower reaction solution of ice bath (I) adds in reaction solution (II), under condition of ice bath, stirs 1h, then stirring at room 12h, TLC (ethyl acetate/petroleum ether=2: 1) show that Boc-Asp disappears.Filtering dicyclohexylurea (DCU) (DCU), THF is removed in decompression.Residue 50mL acetic acid ethyl dissolution.The solution obtaining is used saturated NaHCO successively 3the aqueous solution is washed, the saturated NaCl aqueous solution is washed, 5%KHSO 4the aqueous solution is washed with the saturated NaCl aqueous solution and is washed.Organic phase anhydrous Na 2sO 4dry, filtration, filtrate decompression are concentrated into dry, obtain 1.6g (73.0%) the present embodiment title compound, are beige oily compound.ESI-MS(m/e)：458[M+H] +；
Embodiment 2 preparation Boc-Asp[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3(1b)
According to the preparation method of embodiment 1 by 0.6g (2.5mmol) Boc-Asp and 0.1g (6.1mmol) CH 3(CH 2) 8cH 2oH makes 1.2g (90.2%) the present embodiment title compound, is colorless oil.ESI-MS(m/e)：514[M+H] +；
Embodiment 3 preparation Boc-Asp[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3(1c)
According to the method for embodiment 1 by 2.3g (10.0mmol) Boc-Asp and 4.1g (24.0mmol) CH 3(CH 2) 10cH 2oH makes 5.0g (87.7%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：570[M+H] +；
Embodiment 4 preparation Boc-Asp[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3(1d)
According to the method for embodiment 1 by 2.3g (10.0mmol) Boc-Asp and 5.0g (23.3mmol) CH 3(CH 2) 6cH 2oH makes 5.2g (83.0%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：626[M+H] +；
Embodiment 5 preparation Boc-Asp[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3(1e)
According to the method for embodiment 1 by 5.3g (22.9mmol) Boc-Asp and 13.30g (54.90mmol) CH 3(CH 2) 6cH 2oH makes 14.30g (91.70%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：682[M+H] +；
Embodiment 6 preparation Boc-Asp[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3(1f)
According to the method for embodiment 1 by 5.4g (23.1mmol) Boc-Asp and 15.0g (55.6mmol) CH 3(CH 2) 16cH 2oH makes 15.6g (91.5%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：738[M+H] +；
Embodiment 7 preparation HClAsp[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3(2a)
By 1.60g (3.50mmol) Boc-Asp[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3be dissolved in 25ml4mol/L hydrogenchloride-ethyl acetate solution, stirring at room 2 hours, TLC (ethyl acetate/petroleum ether=1: 2) show that raw material point disappears, concentrating under reduced pressure is removed ethyl acetate, and residue repeatedly adds a small amount of ether and carries out concentrating under reduced pressure to remove hydrogen chloride gas.Finally adding a small amount of ether residue is ground to form to 1.8g (95.2%) the present embodiment title compound, is colorless solid powder, is directly used in next step reaction.ESI-MS(m/e)：394[M+H] +；
Embodiment 8 preparation HClAsp[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3(2b)
According to the method for embodiment 7, from 3.00g (5.85mmol) Boc-Asp[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3making 2.1g (80.99%) the present embodiment title compound, is beige pressed powder.ESI-MS(m/e)：450[M+H] +；
Embodiment 9 preparation HClAsp[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3(2c)
According to the method for embodiment 7, from 1.8g (5.0mmol) Boc-Asp[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3making 2.3g (92.1%) the present embodiment title compound, is beige pressed powder.ESI-MS(m/e)：506[M+H] +。
Embodiment 10 preparation HClAsp[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3(2d)
According to the method for embodiment 7, from 0.6g (1.0mmol) Boc-Asp[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3making 0.5g (89.2%) the present embodiment title compound, is beige pressed powder.ESI-MS(m/e)：562[M+H] +。
Embodiment 11 preparation HClAsp[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3(2e)
According to the method for embodiment 7, from 0.6g (1.0mmol) Boc-Asp[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3making 0.6g (96.7%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：618[M+H] +；
Embodiment 12 preparation HClAsp[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3(2f)
According to the method for embodiment 7, from 0.7g (1.0mmol) Boc-Asp[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3making 0.5g (88.6%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：674[M+H] +；
Embodiment 13 preparation Boc-Lys (Z)-Asp[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3(3a)
According to the method for embodiment 1 by 2.8g (7.3mmol) Boc-Lys (Z) and 2.6g (6.6mmol) HClAsp[OCH 2(CH 2) 6cH]-OCH 2(CH 2) 6cH 3making 1.2g (26.8%) the present embodiment title compound, is colorless oil.ESI-MS(m/e)：720[M+H] +，
Figure BDA0000064434720000061
Embodiment 14 preparation Boc-Lys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3(3b)
According to the method for embodiment 1 by 1.9g (5.0mmol) Boc-Lys (Z) and 2.6g (6.6mmol) HClAsp[OCH 2(CH 2) 8cH]-OCH 2(CH 2) 8cH 3making 3.0g (78.6%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：776[M+H] +，
Figure BDA0000064434720000062
Embodiment 15 preparation Boc-Lys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3(3c)
According to the method for embodiment 1 by 2.4g (6.5mmol) Boc-Lys (Z) and 3.0g (5.9mmol) HClAsp[OCH 2(CH 2) 10cH]-OCH 2(CH 2) 10cH 3making 2.9g (59.7%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：832[M+H] +，
Figure BDA0000064434720000063
Embodiment 16 preparation Boc-Lys (Z)-Asp-[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3(3d)
According to the method for embodiment 1 by 1.3g (3.5mmol) Boc-Lys (Z) and 2.0g (3.5mmol) HClAsp[OCH 2(CH 2) 12cH]-OCH 2(CH 2) 12cH 3making 1.5g (48.3%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：888[M+H] +，
Figure BDA0000064434720000064
Embodiment 17 preparation Boc-Lys (Z)-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3(3e)
According to the method for embodiment 1 by 0.4g (1.2mmol) Boc-Lys (Z) and 0.6g (0.9mmol) HClAsp[OCH 2(CH 2) 14cH]-OCH 2(CH 2) 14cH 3making 5.6g (40.0%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：944[M+H] +，
Figure BDA0000064434720000065
Embodiment 18 preparation Boc-Lys (Z)-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3(3f)
According to the method for embodiment 1 by 0.4g (1.1mmol) Boc-Lys (Z) and 0.6g (0.8mmol) HClAsp[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3making 2.6g (29.2%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：1000[M+H] +，
Figure BDA0000064434720000071
Embodiment 19 preparation HClLys (Z)-Asp-[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3(4a)
According to the method for embodiment 7, from 1.2g (1.7mmol) Boc-Lys (Z)-Asp-[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3making 1.0g (86.3%) the present embodiment title compound, is beige oily matter.ESI-MS(m/e)：656[M+H] +，
Figure BDA0000064434720000072
Figure BDA0000064434720000073
Embodiment 20 preparation HClLys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3(4b)
According to the method for embodiment 7, from 2.0g (2.5mmol) Boc-Lys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3making 1.5g (81.5%) the present embodiment title compound, is beige oily matter.ESI-MS(m/e)：656[M+H] +，
Figure BDA0000064434720000074
Figure BDA0000064434720000075
Embodiment 21 preparation HClLys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3(4c)
According to the method for embodiment 7, from 3.1g (3.7mmol) Boc-Lys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3making 2.4g (88.9%) the present embodiment title compound, is beige oily matter.ESI-MS(m/e)：712[M+H] +，
Figure BDA0000064434720000077
Embodiment 22 preparation HClLys (Z)-Asp-[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3(4d)
According to the method for embodiment 7, from 3.0g (3.3mmol) Boc-Lys (Z)-Asp-[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3making 2.6g (92.9%) the present embodiment title compound, is beige oily matter.ESI-MS(m/e)：768[M+H] +，
Figure BDA0000064434720000078
Figure BDA0000064434720000079
Embodiment 23 preparation HClLys (Z)-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3(4e)
According to the method for embodiment 7, from 3.7g (3.9mmol) Boc-Lys (Z)-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3making 3.3g (97.1%) the present embodiment title compound, is beige oily matter.ESI-MS(m/e)：768[M+H] +，
Embodiment 24 preparation HClLys (Z)-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3(4f)
According to the method for embodiment 7, from 2.5g (2.5mmol) Boc-Lys (Z)-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3making 2.3g (98.4%) the present embodiment title compound, is beige oily matter.ESI-MS(m/e)：824[M+H] +，
Figure BDA0000064434720000081
Figure BDA0000064434720000082
Embodiment 25 preparation Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3(5a)
According to the method for embodiment 1 by 1.4g (8.3mmol) Boc-Gly and 6.5g (9.8mmol) HClLys (Z)-Asp-[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3making 6.3g (80.0%) the present embodiment title compound, is beige oily matter.ESI-MS(m/e)：776[M+H] +，
Figure BDA0000064434720000083
Figure BDA0000064434720000084
Embodiment 26 preparation Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3(5b)
According to the method for embodiment 1 by 1.0g (5.4mmol) Boc-Gly and 3.5g (4.8mmol) HClLys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3making 3.8g (92.6%) the present embodiment title compound, is beige oily matter.ESI-MS(m/e)：832[M+H] +，
Figure BDA0000064434720000085
Figure BDA0000064434720000086
Embodiment 27 preparation Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3(5c)
According to the method for embodiment 1 by 1.1g (6.1mmol) Boc-Gly and 5.6g (7.3mmol) HClLys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3making 5.0g (92.6%) the present embodiment title compound, is faint yellow solid.ESI-MS(m/e)：888[M+H] +，
Figure BDA0000064434720000087
Embodiment 28 preparation Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3(5d)
According to the method for embodiment 1 by 1.4g (8.0mmol) Boc-Gly and 6.0g (7.2mmol) HClLys (Z)-Asp-[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3making 5.0g (77.9%) the present embodiment title compound, is faint yellow solid.ESI-MS(m/e)：944[M+H] +，
Figure BDA0000064434720000089
Figure BDA00000644347200000810
Embodiment 29 preparation Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3(5e)
According to the method for embodiment 1 by 0.5g (2.8mmol) Boc-Gly and 3.0g (3.4mmol) HClLys (Z)-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3making 2.5g (91.4%) the present embodiment title compound, is faint yellow solid.ESI-MS(m/e)：1000[M+H] +，
Figure BDA00000644347200000811
Figure BDA00000644347200000812
Embodiment 30 preparation Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3(5f)
According to the method for embodiment 1 by 0.9g (4.8mmol) Boc-Gly and 5.5g (5.8mmol) HClLys (Z)-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3making 4.8g (94.1%) the present embodiment title compound, is faint yellow solid.ESI-MS(m/e)：1056[M+H] +，
Figure BDA0000064434720000091
Figure BDA0000064434720000092
Embodiment 31 preparation HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3(6a)
According to the method for embodiment 7, from 5.2g (6.7mmol) Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3making 4.5g (93.8%) the present embodiment title compound, is beige solid.ESI-MS(m/e)：714[M+H] +，
Embodiment 32 preparation HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3(6b)
According to the method for embodiment 7, from 3.8g (4.6mmol) Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3making 3.4g (97.1%) the present embodiment title compound, is beige solid.ESI-MS(m/e)：770[M+H] +，
Figure BDA0000064434720000095
Figure BDA0000064434720000096
Embodiment 33 preparation HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3(6c)
According to the method for embodiment 7, from 5.0g (5.6mmol) Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3making 4.5g (97.8%) the present embodiment title compound, is beige solid.ESI-MS(m/e)：826[M+H] +，
Figure BDA0000064434720000097
Figure BDA0000064434720000098
Embodiment 34 preparation HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3(6d)
According to the method for embodiment 7, from 5.3g (5.6mmol) Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3making 4.4g (88.8%) the present embodiment title compound, is beige solid.ESI-MS(m/e)：882[M+H] +，
Figure BDA0000064434720000099
Embodiment 35 preparation HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3(6e)
According to the method for embodiment 7, from 2.5g (2.5mmol) Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3making 2.1g (87.5%) the present embodiment title compound, is beige solid.ESI-MS(m/e)：938[M+H] +，
Figure BDA00000644347200000911
Figure BDA00000644347200000912
Embodiment 36 preparation HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3(6f)
According to the method for embodiment 7, from 4.8g (4.5mmol) Boc-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3making 4.0g (88.8%) the present embodiment title compound, is beige solid.ESI-MS(m/e)：994[M+H] +，
Figure BDA0000064434720000101
Embodiment 37
Preparation Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3(7a)
According to the method for embodiment 1 by 1.8g (5.2mmol) Boc-His (Boc) and 4.5g (6.3mmol) HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3make, gained compound obtains 750mg water white transparency oily thing through column chromatography purification, purification condition: sherwood oil: acetone=1: 1, and yield is 14.4%.ESI-MS(m/e)：1014[M+H] +；mp 67-69℃.
Figure BDA0000064434720000103
1H-NMR(CDCl 3，300MHz)：δ/ppm＝8.10(s，1H)，7.94(s，1H)，7.31(m，6H)，7.202(m，2H)，5.75(s，1H)，5.12(s，1H)，5.06(s，2H)，4.80(m，1H)，4.51(m，1H)，4.40(s，1H)，4.03(m，5H)，3.11(s，2H)，2.85(m，3H)，1.25-1.85(m，41H)，0.86(t，J＝6.3Hz，6H).
Embodiment 38
Preparation Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3(7b)
According to the method for embodiment 1 by 1.7g (4.7mmol) Boc-His (Boc) and 3.4g (4.4mmol) HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3make, gained compound obtains 1g water white transparency oily thing through column chromatography purification, purification condition: chloroform: methyl alcohol=100: 1, and yield is 19.6%.ESI-MS(m/e)：1071[M+H] +；mp 66-67℃.
Figure BDA0000064434720000104
1H-NMR(CDCl 3，300MHz)：δ/ppm＝8.35(m，1H)，8.03(m，1H)，7.24(m，6H)，4.99(s，2H)，4.62(t，J＝6.6Hz，1H)，4.28(m，1H)，4.21(m，1H)，4.00(t，J＝6Hz，4H)，3.710(m，2H)，3.36(d，6H)，2.95-2.66(m，6H)，1.83-1.15(m，54H)，0.848(t，J＝5.7Hz，6H).
Embodiment 39
Preparation Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3(7c)
According to the method for embodiment 1 by 1.6g (4.5mmol) Boc-His (Boc) and 4.5g (5.5mmol) HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3make, gained compound obtains 800mg water white transparency oily thing through column chromatography purification, purification condition: oil mystery: acetone=1: 1, and yield is 15.9%.ESI-MS(m/e)：1149[M+H] +；mp 69-70℃.
Figure BDA0000064434720000105
1H-NMR(CDCl 3，300MHz)：δ/ppm＝8.21(s，1H)，7.79s，1H)，7.36-7.20(m，6H)，5.34(s，1H)，5.11(s，2H)，4.84(m，1H)，4.50(m，2H)，4.08(m，6H)，3.19(d，J＝6Hz，3H)，3.03(m，1H)，2.83(m，1H)，1.702-1.27(m，67H)，0.87(t，J＝5.7Hz，6H).
Embodiment 40
Preparation Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3(7d)
According to the method for embodiment 1 by 1.6g (7.3mmol) Boc-His (Boc) and 5.4g (6.1mmol) HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3make, gained compound obtains 1.4g colorless solid through column chromatography purification, purification condition: chloroform: methyl alcohol=100: 1, and yield is 19.4%.ESI-MS(m/e)：1183[M+H] +；mp 68-69℃.
Figure BDA0000064434720000111
1H-NMR(CDCl 3，300MHz)：δ/ppm＝8.16(m，1H)，7.95(s，1H)，7.36-7.17(m，6H)，7.09(d，J＝8.7Hz，1H)，5.72(s，1H)，5.45(s，1H)5.08(m，1H)，4.82(m，1H)，4.50(m，1H)，4.42(s，1H)，4.11(m，2H)，3.99(s，1H)，3.16(m，2H)，2.99-2.76(m，3H)，1.89(m，1H)，1.48-1.25(m，45H)，0.85(t，J＝6.6Hz，6H).
Embodiment 41
Preparation Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3(7e)
According to the method for embodiment 1 by 0.9g (2.7mmol) Boc-His (Boc) and 3.0g (3.2mmol) HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3make, gained compound obtains 500mg colorless solid through column chromatography purification, purification condition: sherwood oil: acetone=1: 1, and yield is 15.1%.ESI-MS(m/e)：1240[M+H] +；mp 66-67℃.
Figure BDA0000064434720000112
1H-NMR(CDCl 3，300MHz)：δ/ppm＝8.15(s，1H)，7.41-7.05(m，6H)，5.72(s，1H)，5.38(s，1H)，5.14(s，2H)，4.82(m，1H)，4.54(m，1H)，4.42(s，1H)，4.14-3.87(m，6H)，3.17(d，J＝6Hz，3H)，2.99-2.76(m，3H)，1.90-1.18(m，75H)，0.87(t，J＝5.7Hz，6H).
Embodiment 42
Preparation Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3(7f)
According to the method for embodiment 1 by 1.2g (3.4mmol) Boc-His (Boc) and 4.0g (4.0mmol) HClGly-Lys (Z)-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3make, gained compound obtains 500mg colorless solid through column chromatography purification, purification condition: sherwood oil: acetone=1: 1, and yield is 11.3%.ESI-MS(m/e)：1295[M+H] +；mp 71-73℃.
Figure BDA0000064434720000113
1H-NMR(CDCl 3，300MHz)：δ/ppm＝8.16(s，1H)，7.85(s，1H)，7.36-7.06(m，7H)，5.72(s，1H)，5.38(s，1H)，5.10(s，2H)，4.82(m，1H)，4.51(m，1H)，4.42(m，1H)，4.08(m，6H)，3.48(m，1H)，3.17(d，J＝6Hz，3H)，2.99-2.77(m，3H)，2.01(m，3H)，1.91-1.19(m，87H)，0.87(t，J＝5.7Hz，6H).
Embodiment 43
Preparation HClHis-Gly-Lys-Asp-[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3(9a)
According to the method for embodiment 7, from 200mg (0.2mmol) Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3make HClHis-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 6cH 3]-OCH 2(CH 2) 6cH 3, by gained compound be placed in 100ml eggplant-shape bottle, with dissolve with ethanol, add appropriate Pd/C (approximately 20%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dry.Making 93.6mg (70%) the present embodiment title compound, is brown solid.ESI-MS(m/e)：680[M+H] +；mp 66-68℃.
Figure BDA0000064434720000121
Figure BDA0000064434720000122
1H-NMR(DMSO-d 6，300MHz)：δ/ppm＝8.92(s，1H)，8.45(m，2H)，8.15(s，1H)，7.67(m，1H)，6.97(m，1H)，4.64(m，1H)，4.32(s，2H)，3.89(m，5H)，3.39(m，2H)，3.06(m，2H)，2.75(m，4H)，1.55(m，8H)，1.43-1.18(m，25H)，0.86(t，J＝6.3Hz，6H).
Embodiment 44
Preparation HClHis-Gly-Lys-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3(9b)
According to the method for embodiment 7, from 200mg (0.2mmol) Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3make HClHis-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 8cH 3]-OCH 2(CH 2) 8cH 3, by gained compound be placed in 100ml eggplant-shape bottle, with dissolve with ethanol, add appropriate Pd/C (approximately 20%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dry.Making 97.7mg (71%) the present embodiment title compound, is brown solid.ESI-MS(m/e)：737[M+H] +；mp 66-67℃.
Figure BDA0000064434720000123
1H-NMR(DMSO-d 6，300MHz)：δ/ppm＝8.89(s，1H)，8.47(m，1H)，8.18(s，1H)，7.66(s，1H)，6.96(s，1H)，4.64(m 1H)，4.28(m，1H)，4.32(m，1H)，4.00(m，4H)，3.05(m，2H)，2.73(m，4H)，1.65-1.13(m，52H)，0.84(t，J＝5.7Hz，6H).
Embodiment 45
Preparation HClHis-Gly-Lys-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3(9c)
According to the method for embodiment 7, from 200mg (0.2mmol) Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3make HClHis-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 10cH 3]-OCH 2(CH 2) 10cH 3, by gained compound be placed in 100ml eggplant-shape bottle, with dissolve with ethanol, add appropriate Pd/C (approximately 20%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dry.Making 88.5mg (65%) the present embodiment title compound, is brown solid.ESI-MS(m/e)：793[M+H] +；mp 69-70℃.
Figure BDA0000064434720000131
Figure BDA0000064434720000132
1H-NMR(DMSO-d 6，300MHz)：δ/ppm＝8.61(m，1H)，8.42(m，1H)，8.22(m，1H)，7.54(s，1H)，4.62(m，1H)，4.31(m，2H)，4.01(m，5H)，3.81(m，1H)，2.72(m，4H)，1.53(m，8H)，1.34(m，2H)，1.22-1.45(m，36H)，0.84(t，J＝5.7Hz，6H).
Embodiment 46
Preparation HClHis-Gly-Lys-Asp-[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3(9d)
According to the method for embodiment 7, from 200mg (0.2mmol) Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3make HClHis-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 12cH 3]-OCH 2(CH 2) 12cH 3, by gained compound be placed in 100ml eggplant-shape bottle, with dissolve with ethanol, add appropriate Pd/C (approximately 20%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dry.Making 100.3mg (70%) the present embodiment title compound, is brown solid.ESI-MS(m/e)：849[M+H] +；mp 66-67℃.
Figure BDA0000064434720000133
1H-NMR(DMSO-d 6，300MHz)：δ/ppm＝9.05(m，1H)，8.55(m，2H)，8.42(m，2H)，8.07(s，1H)，7.34(m，3H)，4.64(m，1H)，4.29(m，2H)，4.00(m，6H)，2.85(m，5H)，1.68-1.22(m，56H)，0.85(t，J＝5.7Hz，6H).
Embodiment 47
Preparation HClHis-Gly-Lys-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3(9e)
According to the method for embodiment 7, from 200mg (0.2mmol) Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3make HClHis-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 14cH 3]-OCH 2(CH 2) 14cH 3, by gained compound be placed in 100ml eggplant-shape bottle, with dissolve with ethanol, add appropriate Pd/C (approximately 20%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dry.Making 100.4mg (69%) the present embodiment title compound, is brown solid.ESI-MS(m/e)：905[M+H] +；mp 68-69℃.
Figure BDA0000064434720000136
1H-NMR(DMSO-d 6，300MHz)：δ/ppm＝9.05(m，1H)，8.45(m，2H)，8.26(m，1H)，8.14(s，1H)，7.88(m，3H)，6.99(m，1H)，4.62(m，2H)，4.33(m，1H)，4.02(m，6H)，2.75(m，4H)，1.84(m，1H)，1.54(m，7H)，1.39-1.18(m，52H)，0.85(t，J＝5.7Hz，6H).
Embodiment 48
Preparation HClHis-Gly-Lys-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3(9f)
According to the method for embodiment 7, from 200mg (0.2mmol) Boc-His (Boc)-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3make HClHis-Gly-Lys (Z)-Asp-[OCH 2(CH 2) 16cH 3]-OCH 2(CH 2) 16cH 3, by gained compound be placed in 100ml eggplant-shape bottle, with dissolve with ethanol, add appropriate Pd/C (approximately 20%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dry.Making 74.4mg (50%) the present embodiment title compound, is brown solid.ESI-MS(m/e)：961[M+H] +；mp 66-67℃.
Figure BDA0000064434720000141
Figure BDA0000064434720000142
1H-NMR(DMSO-d 6，300MHz)：δ/ppm＝9.08(m，2H)，8.45(m，4H)，8.39(m，1H)，8.03(s，3H)，7.53(s，1H)，7.26(m，1H)，4.62(m，1H)，4.33(m，2H)，4.00(m，7H)，2.66(m，4H)，1.91(m，4H)，1.54(m，8H)，1.24-1.19(m，57H)，0.85(t，J＝5.7Hz，6H).
The restraining effect of 49 pairs of saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters of embodiment to mouse spleen lymphocyte mitogen propagation
De-neck is put to death mouse, and the aseptic spleen of getting grinds with 200 order steel meshes and piston, with PBS liquid, washes under twice, 1500 rev/min of condition centrifugal 10 minutes, counts with complete RPMI-1640 nutrient solution, to be made into splenocyte 5 * 10 afterwards 6the cell suspension of/mL, (every sky is containing 5 * 10 in 96 well culture plates to add 100 μ L cell suspensions 5individual cell).Every hole adds 20 μ L canavalines (ConA final concentration is 5 μ g/mL), and it is 0.05 CO that this 96 porocyte culture plate is placed in volume fraction 2in the incubator of saturated humidity, cultivate 4h for 37 ℃.After 4h by default concentration gradient add to be measured, through the sample (1 * 10 of sterilising treatment -4, 8 * 10 -5, 5 * 10 -5, 2 * 10 -5, 1 * 10 -5, 8 * 10 -6, 5 * 10 -6, 1 * 10 -6), 3 multiple holes of each concentration, control group adds the solvent of isopyknic sample dissolution.Establish not containing compound control wells with only containing the cell blank hole without ConA with amount nutrient solution simultaneously.(n=3) all repeated in each hole 3 times.After cultivating 48h, use the restraining effect of mtt assay detection compound to splenic lymphocyte.
Restraining effect according to the compound of formula " inhibiting rate=(D contrast-D pastille)/D contrast * 100% " calculating different concns to spleen lymphocyte proliferation, according to the concentration of cell relative survival rate and compound, draw cell growth curve, utilize this growth curve try to achieve half inhibiting rate ( dxiC 50).Result is listed table 1 in, and result shows that of the present invention pair of saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide ester has clear and definite restraining effect to mice spleen lymphocytes proliferation.
The restraining effect of table 19a-f to mouse spleen lymphocyte mitogen propagation
Note: CsA is S-Neoral n=3
The restraining effect of 50 pairs of saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters of embodiment to macrophage phagocytic
Growth conditions is good, in the Ana-1 of logarithmic phase mouse macrophage with 1 * 10 5the density of individual/mL is inoculated in 96 orifice plates, every hole 100 μ L, 37 ℃, 5%CO 2in incubator, cultivate 4 hours, by default concentration gradient add to be measured, through the sample (1 * 10 of sterilising treatment -4, 8 * 10 -5, 5 * 10 -5, 2 * 10 -5, 1 * 10 -5, 8 * 10 -6, 5 * 10 -6, 1 * 10 -6), 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 50 μ L 0.1%, is placed in 37 ℃ and hatches 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, adds cytolysate (ethanol: acetic acid=1: 1) 50 μ L, 4 ℃ are spent the night, microplate reader detection absorbance, wavelength 540nm.(n=3) all repeated in each hole 3 times.
Restraining effect according to the compound of formula " inhibiting rate=(D contrast-D pastille)/D contrast * 100% " calculating different concns to the phagocytic activity of scavenger cell, according to the concentration of cell relative survival rate and compound, draw cell growth curve, utilize this growth curve try to achieve half inhibiting rate ( dxiC50).Result is listed table 2 in, and result shows that of the present invention pair of saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide ester engulf and have clear and definite restraining effect Ana-1 mouse macrophage.
The restraining effect that table 29a-f engulfs mouse macrophage
Figure BDA0000064434720000152
Figure BDA0000064434720000161
Note: CsA is S-Neoral, n=3
The autonomous dress performance evaluation of embodiment 519a-f
1) particle diameter (unit: nm) of 9a-f nanometer ball in the aqueous solution
Two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters are configured to the aqueous solution of 1 μ mol/mL, on laser nano particle size analyzer, measure particle diameter for 25 ℃.METHOD FOR CONTINUOUS DETERMINATION 8 days, records its particle diameter.Result is listed table 3 in.Data show, all can self-assembly granulating in of the present invention pair of saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide aqueous solution of ester through being stabilized in the nanometer ball of 200-600nm.
The nanometer ball particle diameter of table 39a-f self-assembly in water
2) form of the nanometer ball of 9a-f
It is 1 * 10 that 9a-f is made into concentration -12the aqueous solution of mg/mL, then drops in this solution on copper mesh, observes the form of nanometer ball after the dry solvent that volatilizees under JEM-1230 transmission electron microscope.Mensuration shows, the nanometer ball of 9a-f formation rule.The transmission electron microscope photo of 9d, as representative, is described with Fig. 2.
The employing mouse ear rear myocardium tissue transplantation experiments of embodiment 529a-f
Acceptor mouse is through 10% urethane intraperitoneal injection of anesthesia.1% bromogeramine tincture auricle partly sterilised, shaves hair, holds eye scissors 1/3 place before auricle dorsal part center line and makes an otch of locating do a 3-4 millimeters long vertical with auricle center line, does not damage auricle vein.Hold tweezers to have sharp ears direction blunt separation subcutis, make it into a tube chamber.New life is placed in to trash ice 75% alcohol skin degerming after a minute for mouse, cuts open chest and win heart.Heart is placed in without beating 1-2 time in PBS liquid with blood more than the emptying chambers of the heart.During transplanting, with blade, longitudinally cuing open for the heart, to become basic grade large 2 half, and myofiber becomes an inclined-plane.Cardiac muscular tissue is transplanted and inserted in acceptor mouse ear chamber, skin incision is sewed up to a pin.(isolated time of cardiac muscular tissue is no more than 2 minutes) is local with finger flicking, makes graft and be adjacent to by the surrounding tissue of mouse.Post-transplantation administration on the same day, every day intraperitoneal injection 0.2mL.
Within postoperative the 6th day, play the electrocardiosignal that records heart transplantation muscular tissue every day.During test ectocardia electrograph, positive and negative electrode is placed in respectively heart transplant both sides, and earthing pole is connected to mouse hind leg.
Positive reference substance cyclosporin A is pressed dosage and the CMCNa (0.5%) of 1.0 μ mol/kg/day, intraperitoneal administration by the dosage of 2.5 μ mol/kg/day, compound 9a-f.Once a day, 0.2mL/ mouse, successive administration 15 days, administration is 15 times altogether.
Within postoperative 15 days, finish to observe.Statistics prolonged Survival of Rat Cardiac.This experimental data statistics all adopts t check and variance analysis, with
Figure BDA0000064434720000171
represent.
Table 4 mouse ear rear myocardium tissue's survival time
Note: n=12, the survival time is used
Figure BDA0000064434720000173
it represents, survival rate use
Figure BDA0000064434720000174
% represents, the positive contrast S-Neoral of CsA;
A) compare P < 0.01 with NS; B) compare P < 0.05 with NS;
The employing mouse ear rear myocardium tissue transplantation experiments dose-effect relationship of embodiment 539a-f
By the method for embodiment 52, choose the dose-effect relationship that 10.0 μ mol/kg/day, 1.0 μ mol/kg/day and 0.01 μ mol/kg/day Three doses are investigated 9f.Result shows, the mouse ear rear myocardium tissue survival time use show dose dependency (table 5) of 9f.
The impact of table 5 various dose 9eDui mouse ear rear myocardium tissue's survival time
Figure BDA0000064434720000181
Note: n=12, the survival time is used
Figure BDA0000064434720000182
it represents, survival rate use
Figure BDA0000064434720000183
% represents, the positive contrast S-Neoral of CsA; A) compare P < 0.01 with NS group, compare P < 0.05 with 0.01 μ mol/kg group; B) compare P < 0.01 with NS and 0.01 μ mol/kg, compare P < 0.05 with 1.0 μ mol/kg groups; C) compare P > 0.05. with NS group
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, revise, and even equivalence change, but all will fall within the scope of protection of the present invention.

Claims (5)

1. 6 of general formula I kinds of two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters with immunosuppressive activity:
His-Gly-Lys-Asp[OCH 2(CH 2)nCH 3]-OCH 2(CH 2)nCH 3 I
Wherein, the n=6 in general formula I, 8,10,12,14 or 16.
2. a method of preparing two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters of claim 1, is characterized in that, comprises the following steps:
(1) at (Boc) 2o and NaOH are converted into N-t-butoxycarbonyl glycine by glycine under existing;
(2) at (Boc) 2o and NaOH are converted into N-tertbutyloxycarbonyl aspartic acid by aspartic acid under existing;
(3) at (Boc) 2o and triethylamine are converted into N-tertbutyloxycarbonyl-N by Histidine under existing im-tertbutyloxycarbonyl-Histidine;
(4) under dicyclohexyl carbonyl diimine, N-hydroxy benzo triazole, anhydrous tetrahydro furan exist by the saturated fatty alcohol condensation of N-tertbutyloxycarbonyl aspartic acid and doubling dose, generate pair saturated fatty alcohol N-tertbutyloxycarbonyl aspartates;
(5) in hydrogenchloride-ethyl acetate, two saturated fatty alcohol N-tertbutyloxycarbonyl aspartates are removed to tertiary butyloxycarbonyl protecting group, generate two saturated fatty alcohol aspartates;
(4) under dicyclohexyl carbonyl diimine, N-hydroxy benzo triazole, anhydrous tetrahydro furan exist by N α-tertbutyloxycarbonyl-N ε-carbobenzoxy-(Cbz) Methionin and two saturated fatty alcohol aspartic acid ester condensations, generate two saturated fatty alcohol N α-tertbutyloxycarbonyl-N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(6) in hydrogenchloride-ethyl acetate by two saturated fatty alcohol N α-tertbutyloxycarbonyl-N ε-carbobenzoxy-(Cbz) lysyl aspartate removes tertiary butyloxycarbonyl protecting group, generates two saturated fatty alcohol N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(7) in anhydrous tetrahydro furan, under dicyclohexyl carbonyl diimine and N-hydroxy benzo triazole exist by two saturated fatty alcohol N ε-carbobenzoxy-(Cbz) lysyl aspartate and the condensation of N-t-butoxycarbonyl glycine, generate two saturated fatty alcohol N-tertbutyloxycarbonyl glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(8) under hydrogenchloride-ethyl acetate exists by two saturated fatty alcohol N-tertbutyloxycarbonyl glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate removes tertbutyloxycarbonyl, generates two saturated fatty alcohol glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(9) in anhydrous tetrahydro furan, under dicyclohexyl carbonyl diimine and N-hydroxy benzo triazole exist by two saturated fatty alcohol glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate and N-tertbutyloxycarbonyl-N imthe condensation of-tertbutyloxycarbonyl-Histidine, generates two saturated fatty alcohol N-tertbutyloxycarbonyl-N im-tertbutyloxycarbonyl histidyl-glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(10) in hydrogenchloride-ethyl acetate by two saturated fatty alcohol N-tertbutyloxycarbonyl-N im-tertbutyloxycarbonyl histidyl-glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate removes tertbutyloxycarbonyl, generates two saturated fatty alcohol histidyl-glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate;
(11) in dehydrated alcohol, under Pd/C exists by two saturated fatty alcohol histidyl-glycyl N ε-carbobenzoxy-(Cbz) lysyl aspartate hydrogenolysis, generates two saturated fatty alcohol histidyl-glycyl lysyl aspartates.
3. the purposes of 6 kinds of two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters with immunosuppressive activity claimed in claim 1 in preparation immunosuppressor.
4. the purposes of 6 kinds of two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters with immunosuppressive activity claimed in claim 1 in preparation immunosuppression pharmacome.
5. the purposes of 6 kinds of two saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters with immunosuppressive activity claimed in claim 1 in preparing micro emulsion or liposome targeted drug.
CN201110141197.7A 2011-05-27 2011-05-27 Double saturated aliphatic chain alcohol His-Gly-Lys-Asp tetrapeptide esters and preparation method and application thereof Expired - Fee Related CN102796170B (en)

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