CN104211761B - Conjugates of saturated aliphatic chain alcohol, dexamethasone, and His-Gly-Glu, preparation, nano structure, and applications thereof - Google Patents
Conjugates of saturated aliphatic chain alcohol, dexamethasone, and His-Gly-Glu, preparation, nano structure, and applications thereof Download PDFInfo
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Abstract
The invention relates to conjugates of saturated aliphatic chain alcohol, dexamethasone, and His-Gly-Glu, preparation, a nano structure, and applications thereof. The invention discloses 6 saturated aliphatic chain alcohol modified dexamethasone-His-Gly-Glu conjugates represented by the formula 10 a-f, wherein in the formula the n represents 7, 9, 11, 13, 15, or 17. The invention also discloses a preparation method, a nano structure, immunity inhibition activity, inflammation inhibition activity, and pain relieving activity of the conjugates. The invention also finds that the conjugates do not have any side effect leading to osteoporosis. The researches on the inhibition effect of the conjugates on splenic lymphocyte mitogen proliferation reactions and the survival time after mouse retroauricular cardiac muscle transplant show that the conjugates have an excellent immunity inhibition effect. The researches on the effect of the conjugates on the swelling degree of mouse ears which are inflamed due to xylene show that the conjugates have an excellent anti-inflammation effect. The researches on the effect of the conjugates on mouse heat radiation tail flick time show that the conjugates have an excellent pain-relieving effect. The researches on the effect of the conjugates on the mouse thigh bones show that the conjugates cannot cause osteoporosis. So the conjugates have a wide application prospect in the preparation of immunity inhibition drugs.
Description
Technical field
Dexamethasone-the His-Gly-Glu of the 6 kinds of saturated aliphatic chain alcohols modification represented the present invention relates to formula 10a-f is conjugated
Thing, the n=7 in saturated aliphatic chain alcohol, 9,11,13,15,17, it is related to their preparation method, it is related to their nanostructured,
It is related to their immunosupress, anti-inflammatory and analgesic activities, is related to their osteoporosis side effect.The present invention passes through 10a-f pairs
The inhibitory action of SPL mitogen breeder reaction and to the time-to-live after myocardium transplantation after mouse ear, shows them
With outstanding immunosuppressive action, research 10a-f shows that they have outstanding to the scorching ear swelling degree of mouse caused by dimethylbenzene xylene
Antiinflammatory action, influences of the research 10a-f to the mouse heat radiation whipping time, shows that they have outstanding analgesic activity, studies
Influences of the 10a-f to mouse femur, shows that they do not have osteoporosis side effect.Thus 10a-f is preparing immunosuppressive drug
In be with a wide range of applications.The invention belongs to biomedicine field.
Background technology
Diseased organ is substituted with normal organ to be yearned for by clinic always.In transfer operation before 20th century, except cornea
Outside transfer operation, mostly end in failure.1945, the medical biotechnology scholar Peter plum Davao of Britain was via dermatoplastic
Experiment model inquires into the mechanism of repulsion, it is indicated that transfer operation failure is because the repulsion that causes of immunocyte.Find within 1949
Cortex hormone of aadrenaline, the success rate that makes transfer operation using cortex hormone of aadrenaline is greatly improved, immune so as to start
Inhibitor is applied to the beginning of transfer operation.The sequential uses such as immunodepressant such as ciclosporin A and FK506, make transplant organ
Time-to-live further extends.Because these medicines exist degradable, bioavilability is not high, and toxic and side effect is strong, treatment target
The shortcomings of limiting to scheme, so research neotype immunosuppressant is constantly subjected to pay attention to.It was recognized by the inventor that glucocorticoid one
Aspect has strong immunosuppressive activity, on the one hand with osteoporosis side effect.Inventor is also to be recognized that His-Gly-Glu tri-
On the one hand peptide has suppressor activity peptide, on the one hand with the effect for suppressing bone loss.Inventor is also to be recognized that sugared cortex
Hormone and His-Gly-Glu tripeptides covalent bonds, can not only improve immunosuppressive activity, and can eliminate the secondary work of bone loss
With.Inventor it is further recognized that by glucocorticoid, His-Gly-Glu tripeptides and saturated aliphatic chain three's covalent bond, no
Immunosuppressive activity can only be strengthened and the side effect of bone loss is eliminated, nanostructured can be also formed and be conducive to conveying in vivo.According to
These understanding, inventor is proposed glucocorticoid, His-Gly-Glu tripeptides and the covalently bound hair of saturated aliphatic chain three
It is bright.Compared with disclosed immunodepressant, glucocorticoid of the invention, His-Gly-Glu tripeptides and saturated aliphatic chain
The covalently bound immunosupress of three has 5 creativeness of proposition：1) strong immunosuppressive activity；2) the secondary work without bone loss
With；3) form nanostructured and be conducive to conveying in vivo；4) strong anti-inflammatory activity；5) strong analgesic activities.
The content of the invention
First technical problem to be solved by this invention is to provide 6 kinds of formula 10a-f representatives with immunosupress work
Property saturated aliphatic chain alcohol modification dexamethasone-His-Gly-Glu conjugates.N=7 in 10a；N=9 in 10b；N=in 10c
11；N=13 in 10d；N=15 in 10e；N=17 in 10f；
Second technical problem to be solved by this invention is to provide 6 kinds of formula 10a~f with immunosuppressive activity
Saturated aliphatic chain alcohol modification dexamethasone-His-Gly-Glu conjugates preparation method, the method comprises the following steps：
(1) at (Boc)2L-Gly is converted into Boc-Gly in the presence of O and NaOH；
(2) at (Boc)2L-His is converted into Boc-His (Boc) in the presence of O and triethylamine；
(3) in dicyclohexyl carbonyl diimine (DCC), N- hydroxy benzo triazoles (HOBt), anhydrous tetrahydro furan (THF),
By Boc-Glu (OBzl) and saturated fatty alcohol [CH in the presence of N-methylmorpholine (NMM)3(CH2) nOH] condensation, generation saturation Boc-
Glu(OBzl)-O-(CH2)nCH3；
(4) by Boc-Glu (OBzl)-O- (CH in the ethyl acetate solution (4M) of hydrogen chloride2)nCH3Removing Boc, generation
Glu(OBzl)-O-(CH2)nCH3；
(5) in DCC, HOBt, anhydrous THF, by Glu (OBzl)-O- (CH in the presence of NMM2)nCH3With Boc-Gly condensations, life
Into Boc-Gly-Glu (OBzl)-O- (CH2)nCH3；
(6) by Boc-Gly-Glu (OBzl)-O- (CH in the presence of the ethyl acetate solution (4M) of hydrogen chloride2)nCH3Removing
Boc, generation Gly-Glu (OBzl)-O- (CH2)nCH3；
(7) in DCC, HOBt, anhydrous THF, by Gly-Glu (OBzl)-O- (CH in the presence of NMM2)nCH3With Boc-His
(Boc) it is condensed, generation Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)nCH3；
(8) by Boc-His (Boc)-Gly-Glu (OBzl)-O- in the presence of the ethyl acetate solution (4M) of hydrogen chloride
(CH2)nCH3Removing Boc, generation His-Gly-Glu (OBzl)-O- (CH2)nCH3；
(9) by His-Gly-Glu (OBzl)-O- (CH2)nCH3Hydrogenolysis, generation His-Gly-Glu-O- (CH2)nCH3；
(10) at DMAP (DMAP), dexamethasone and succinic anhydride are condensed in the presence of anhydrous THF, generation
Dexamethasone 3- carboxyl propionic esters；
(11) in 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (HClEDC), anhydrous dimethyl base
, be condensed for dexamethasone 3- carboxyls propionic ester and N-hydroxy-succinamide (HOSu) in the presence of NMM, generation by formamide (DMF)
Dexamethasone 3-N- formyl succinyl phosphorons amino propyl acid esters；
(12) by dexamethasone 3-N- formyl succinyl phosphorons amino propyl acid esters and His-Gly- in the presence of dry DMF, NMM
Glu-O-(CH2)nCH3Condensation, generation dexamethasone 3- formyl-His-Gly-Glu-O- (CH2)nCH3Propionic ester.
The 3rd technical problem that the present invention is solved is, by experimental study, evaluates 6 kinds of ground of saturated aliphatic chain alcohol modification
The nanometer property of Sai meter Song-His-Gly-Glu conjugates；
The 4th technical problem that the present invention is solved is, by experimental study, have rated 6 kinds of saturated aliphatic chain alcohol modifications
Dexamethasone-His-Gly-Glu conjugates to mouse spleen lymphocyte to the inhibitory action of the breeder reaction of mitogen；
The 5th technical problem that the present invention is solved is, by experimental study, evaluates what 6 kinds of saturated aliphatic chain alcohols were modified
Dexamethasone-His-Gly-Glu conjugate mouse ears rear myocardium tissue's transplanting survival time；
The 6th technical problem that the present invention is solved is, by experimental study, evaluates what 6 kinds of saturated aliphatic chain alcohols were modified
Dexamethasone-His-Gly-Glu conjugate mouse heat radiation whipping the reaction time；
The 7th technical problem that the present invention is solved is, by experimental study, evaluates what 6 kinds of saturated aliphatic chain alcohols were modified
Dexamethasone-His-Gly-Glu conjugate mouse caused by dimethylbenzene xylene inflammation ear swelling degree；
The 8th technical problem that the present invention is solved is, by experimental study, evaluates what 6 kinds of saturated aliphatic chain alcohols were modified
Influence of the dexamethasone-His-Gly-Glu conjugates to mouse femur.
The present invention is by the dexamethasone-His-Gly-Glu conjugates of the saturated aliphatic chain alcohol modification described in research to spleen
The inhibitory action of lymphocyte mitogen breeder reaction, the influence to the time-to-live after myocardium transplantation after mouse ear is indicated
Compound of the invention has outstanding immunosuppressive action.Before being had a wide range of applications in preparing immune suppressant drug
Scape.
One of purposes of dexamethasone-His-Gly-Glu conjugates of saturated aliphatic chain alcohol modification of the invention is in system
The standby purposes with the steroidal immunodepressant without sclerotin fluffing action；
The two of the purposes of the dexamethasone-His-Gly-Glu conjugates of saturated aliphatic chain alcohol modification of the invention are in system
The standby purposes having concurrently in the immunodepressant of anti-inflammatory and antalgic activity.
Dexamethasone-His-Gly-Glu the conjugates of saturated aliphatic chain alcohol modification of the invention are to prepare micro emulsion or fat
Purposes in plastid targeted drug.
Brief description of the drawings
6 kinds of dexamethasone of the saturated aliphatic chain alcohols modification with immunosuppressive activity that Fig. 1 is represented for formula 10a-f-
The synthetic route of His-Gly-Glu conjugates.I) anhydrous THF, DMAP；Ii) dry DMF, HClEDC；Iii) anhydrous THF,
DMAP, HOBt and NMM；Iv) the ethyl acetate solution (4M) of hydrogen chloride；V) absolute ethyl alcohol, Pd/C and H2；Vi) dry DMF,
NMM；N=7 in 10a, n=9 in 10b, n=11 in 10c, n=13 in 10d, n=15 in 10e, n=17 in 10f.
Fig. 2 is 10a-f 1 × 10-6The representative transmission electron microscope photo of the nanosphere formed in the M aqueous solution.
Specific embodiment
In order to the present invention is expanded on further, a series of embodiments are given below.These embodiments be entirely it is illustrative, it
Only be used for the present invention is specifically described, be not construed as limitation of the present invention.
Embodiment 1 prepares dexamethasone 3- carboxyls propionic ester (1)
3.93g (10mmol) dexamethasone in the presence of 1.46g (12mmol) DMAP (DM AP) with 1.3g
(13mmol) succinic anhydride lucifuge in 150ml tetrahydrofurans (THF) is reacted 48 hours, TLC (CH2Cl2∶CH3OH: HOAC, 20:
1: 0.15) show that raw material is wholly absent.Reactant mixture adds 30ml H2O, is concentrated under reduced pressure, and residue adds KHSO4Regulation pH2, analysis
Colourless powder acetone/the petroleum ether for going out is washed, and obtains 4.64g (94%) title compound, is colourless powder.ES I/MS(m/z)
491[M-H]-。1H NMR (300MHZ, DMSO-d6)：δ/ppm=7.29 (d, J=10.2Hz, 1H), 6.23 (d, J=9.3Hz,
1H), 6.01 (s, 1H), 5.42 (s, 1H), 5.17 (s, 1H), 5.05 (d, J=17.7Hz, 1H), 4.80 (d, J=17.7Hz,
1H), 4.15 (m, 1H), 2.88 (m, 1H), 2.52 (m, 2H), 2.61 (m, 3H), 2.34 (m, 2H), 2.15 (m, 2H), 1.77 (m,
1H), 1.61 (m, 2H), 1.49 (s, 3H), 1.35 (m, 1H), 1.08 (m, 1H), 0.88 (s, 3H), 0.79 (d, J=7.2Hz,
3H)。
Embodiment 2 prepares dexamethasone 3-N- formyl succinyl phosphorons amino propyl acids ester (2)
4.92g (10mmol) dexamethasone succinate (1) is in 2.50g (13mmol) 1- ethyls-(3- dimethylaminos third
Base) exist with 1.50g (13mmol) N-hydroxy-succinamide (HOSu) in the presence of phosphinylidyne diimmonium salt hydrochlorate (HClEDC)
Lucifuge is reacted 24 hours in the solvent of 100ml THF and 10ml DMF, TLC (CH2Cl2∶CH3OH: HOAC, 30: 1: 0.15) show
Raw material is wholly absent.Reactant mixture is concentrated under reduced pressure, and residue adds 150ml ethyl acetate, the solution for obtaining 20ml saturations
NaHCO3The aqueous solution is washed 3 times, is washed 2 times with the 20ml saturation NaCl aqueous solution, with 20ml saturations KHSO4The aqueous solution washes 3 times, Ran Houyong
The 20ml saturation NaCl aqueous solution is washed 2 times, ethyl acetate layer anhydrous sodium sulfate drying, and filtering, filtrate decompression is concentrated to dryness, and is remained
With column chromatography, (petroleum ether: acetone, 1: 2) purifies, obtains 5.37g (91%) title compound, be colourless powder thing.ESI/MS
(m/z)612[M+Na]+。1H NMR (300MHZ, DMSO-d6)：δ/ppm=7.29 (d, J=10.2Hz, 1H), 6.23 (d, J=
10.2Hz, 1H), 6.01 (s, 1H), 5.41 (d, J=3.9Hz, 1H), 5.18 (s, 1H), 5.10 (d, J=17.4Hz, 1H),
4.82 (d, J=17.7Hz, 1H), 4.16 (m, 1H), 2.98 (t, J=6.3Hz, 1H), 2.81 (m, 6H), 2.63 (m, 1H),
2.34 (m, 2H), 2.16 (t, J=11.7Hz, 2H), 1.78 (m, 1H), 1.64 (d, J=11.7Hz, 1H), 1.56 (d, J=
13.5Hz, 1H), 1.50 (s, 3H), 1.34 (m, 1H), 1.08 (m, 1H), 0.89 (s, 3H), 0.80 (d, J=7.2Hz, 3H).
Embodiment 3 prepares Boc-Glu (OBzl)-O- (CH2)7CH3(3a)
1.49g (5mmol) Boc-Glu (OBzl) is dissolved in the anhydrous THF of 20mL, is added in the solution for obtaining under ice bath
0.675g (5mmol) N- hydroxy benzo triazoles (HO Bt), and it is completely dissolved it.1.236g (6mmol) is added after 10 minutes
Dicyclohexyl carbonyl diimine (DCC) obtains reaction solution I.Lower 0.78g (6mmol) CH of ice bath3(CH2)7It is anhydrous that OH is suspended in 20mL
In dichloromethane, it is subsequently adding 1ml N-methylmorpholines (NMM) and adjusts pH9.Stirring 35 minutes, obtains reaction solution II.Ice bath
In the lower II reaction solution I addition reaction solutions, 1h is stirred under first ice bath, then 12h is stirred at room temperature, TLC (ethyl acetate: petroleum ether, 2:
1) display Boc-Gly disappears.Dicyclohexylurea (DCU) (DCU) is filtered, is concentrated under reduced pressure.Residue 50mL ethyl acetate dissolves.Obtain
Solution use saturation NaHCO successively3The aqueous solution is washed, and the saturation NaCl aqueous solution is washed, 5%KHSO4The aqueous solution washes water-soluble with saturation NaCl
Liquid is washed.Ethyl acetate phase anhydrous Na2SO4Dry, filtering, filtrate decompression is concentrated to dryness, and obtains 1.88g (92%) title compound
Thing, is colorless oil.ESI-MS(m/e)：410[M+H]+；
Embodiment 4 prepares Boc-Glu (OBzl)-O- (CH2)9CH3(3b)
According to the preparation method of embodiment 3 by 1.49g (5mmol) Boc-Glu (OBzl) and 0.948g (6mmol) CH3
(CH2)9OH is obtained 1.97g (90.2%) title compound, is colorless oil.ESI-MS(m/e)：438[M+H]+；
Embodiment 5 prepares Boc-Glu (OBzl)-O- (CH2)11CH3(3c)
According to the method for embodiment 3 by 1.49g (5mmol) Boc-Glu (OBzl) and 1.116g (6mmol) CH3(CH2)11OH is obtained 2.04g (87.7%) title compound, is colorless oil.ESI-MS(m/e)：466[M+H]+；
Embodiment 6 prepares Boc-Glu (OBzl)-O- (CH2)13CH3(3d)
According to the method for embodiment 3 by 1.49g (5mmol) Boc-Glu (OBzl) and 1.284g (6mmol) CH3(CH2)13OH is obtained 2.04g (83.0%) title compound, is colorless oil.ESI-MS(m/e)：494[M+H]+；
Embodiment 7 prepares Boc-Glu (OBzl)-O- (CH2)15CH3(3e)
According to the method for embodiment 3 by 1.49g (5mmol) Boc-Glu (OBzl) and 1.452g (6mmol) CH3(CH2)15OH is obtained 2.39g (91.70%) title compound, is colorless oil.ESI-MS(m/e)：522[M+H]+；
Embodiment 8 prepares Boc-Glu (OBzl)-O- (CH2)17CH3(3f)
According to the method for embodiment 3 by 1.49g (5mmol) Boc-Glu (OBzl) and 1.62g (6mmol) CH3(CH2)17OH
2.5g (91.5%) title compound is obtained, is colorless oil.ESI-MS(m/e)：550[M+H]+；
Embodiment 9 prepares HClGlu (OBzl)-O- (CH2)7CH3(4a)
By 1.8g (4.4mmol) Boc-Glu (OBzl)-O- (CH2)7CH3It is dissolved in the acetic acid second of 50ml4mol/L hydrogen chloride
In ester solution (4M) solution, it is stirred at room temperature 2 hours, (ethyl acetate: petroleum ether, 1: 2) shows that raw material point disappears, reaction is mixed to TLC
Compound is concentrated under reduced pressure, and residue adds a small amount of ether to be concentrated under reduced pressure to remove hydrogen chloride gas repeatedly.Finally plus on a small quantity ether will
Residue grinds to form 1.45g (95.2%) title compound, is colourless powder, is directly used in next step reaction.
ESI-MS(m/e)：347.5[M+H]+；
Embodiment 10 prepares HClGlu (OBzl)-O- (CH2)9CH3(4b)
According to the method for embodiment 9, from 1.8g (4.12mmol) Boc-Glu (OBzl)-O- (CH2)9CH31.5g is obtained
(97%) title compound, is colourless powder.ESI-MS(m/e)：375.5[M+H]+；
Embodiment 11 prepares HClGlu (OBzl)-O- (CH2)11CH3(4c)
According to the method for embodiment 9, from 2g (4.3mmol) Boc-Glu (OBzl)-O- (CH2)11CH31.65g is obtained
(96.1%) title compound, is colourless powder.ESI-MS(m/e)：403.5[M+H]+,
Embodiment 12 prepares HClGlu (OBzl)-O- (CH2)13CH3(4d)
According to the method for embodiment 9, from 2g (4.1mmol) Boc-Glu (OBzl)-O- (CH2)13CH31.76g is obtained
(99.5%) title compound, is colourless powder.ESI-MS(m/e)：431.5[M+H]+；
Embodiment 13 prepares HClGlu (OBzl)-O- (CH2)15CH3(4e)
According to the method for embodiment 9, from 2.3g (4.4mmol) Boc-Glu (OBzl)-O- (CH2)15CH31.96g is obtained
(96.7%) title compound, is colourless powder.ESI-MS(m/e)：459.5[M+H]+；
Embodiment 14 prepares HClGlu (OBzl)-O- (CH2)17CH3(4f)
According to the method for embodiment 9, from 2.4g (4.4mmol) Boc-Glu (OBzl)-O- (CH2)17CH32.1g is obtained
(98.6%) title compound, is colourless powder.ESI-MS(m/e)：487.5[M+H]+；
Embodiment 15 prepares Boc-Gly-Glu (OBzl)-O- (CH2)7CH3(5a)
According to the method for embodiment 3 by 0.86g (4.93mmol) Boc-Gly and 1.42g (4.1mmol) HClGlu
(OBzl)-O-(CH2)7CH31.76g (92.2%) title compound is obtained, is colorless oil.ESI-MS(m/e)：467[M+
H]+；
Embodiment 16 prepares Boc-Gly-Glu (OBzl)-O- (CH2)9CH3(5b)
According to the method for embodiment 3 by 0.86g (4.93mmol) Boc-Gly and 1.5g (4.1mmol) HClGlu
(OBzl)-O-(CH2)9CH31.86g (91.7%) title compound is obtained, is colorless oil.ESI-MS(m/e)：495[M+
H]+；
Embodiment 17 prepares Boc-Gly-Glu (OBzl)-O- (CH2)11CH3(5c)
According to the method for embodiment 3 by 0.86g (4.93mmol) Boc-Gly and 1.65g (4.1mmol) HClGlu
(OBzl)-O-(CH2)11CH31.88g (87.8%) title compound is obtained, is colorless oil.ESI-MS(m/e)：523[M+
H]+；
Embodiment 18 prepares Boc-Gly-Glu (OBzl)-O- (CH2)13CH3(5d)
According to the method for embodiment 3 by 0.86g (4.93mmol) Boc-Gly and 1.76g (4.1mmol) HClGlu
(OBzl)-O-(CH2)13CH31.94g (86.0%) title compound is obtained, is colorless oil.ESI-MS(m/e)：551[M+
H]+；
Embodiment 19 prepares Boc-Gly-Glu (OBzl)-O- (CH2)15CH3(5e)
According to the method for embodiment 3 by 0.86g (4.93mmol) Boc-Gly and 1.88g (4.1mmol) HClGlu
(OBzl)-O-(CH2)15CH31.96g (82.6%) title compound is obtained, is colorless oil.ESI-MS(m/e)：579[M+
H]+；
Embodiment 20 prepares Boc-Gly-Glu (OBzl)-O- (CH2)17CH3(5f)
According to the method for embodiment 3 by 0.86g (4.93mmol) Boc-Gly and 1.99g (4.1mmol) HClGlu
(OBzl)-O-(CH2)17CH32.07g (83.4%) title compound is obtained, is colorless oil.ESI-MS(m/e)：607[M+
H]+；
Embodiment 21 prepares HClGly-Glu (OBzl)-O- (CH2)7CH3(6a)
According to the method for embodiment 9, from 1.7g (3.65mmol) Boc-Gly-Glu (OBzl)-O- (CH2)7CH3It is obtained
1.4g (95.4%) title compound, is colourless powder.ESI-MS(m/e)：404.5[M+H]+；
Embodiment 22 prepares HClGly-Glu (OBzl)-O- (CH2)9CH3(6b)
According to the method for embodiment 9, from 1.8g (3.6mmol) Boc-Gly-Glu (OBzl)-O- (CH2)9CH3It is obtained
1.55g (98.4%) title compound, is colourless powder.ESI-MS(m/e)：432.5[M+H]+；
Embodiment 23 prepares HClGly-Glu (OBzl)-O- (CH2)11CH3(6c)
According to the method for embodiment 9, from 1.8g (3.45mmol) Boc-Gly-Glu (OBzl)-O- (CH2)11CH3It is obtained
1.48g (93.5%) title compound, is colourless powder.ESI-MS(m/e)：460.5[M+H]+；
Embodiment 24 prepares HClGly-Glu (OBzl)-O- (CH2)13CH3(6d)
According to the method for embodiment 9, from 1.9g (3.45mmol) Boc-Gly-Glu (OBzl)-O- (CH2)13CH3It is obtained
1.67g (99.6%) title compound, is colourless powder.ESI-MS(m/e)：488.5[M+H]+；
Embodiment 25 prepares HClGly-Glu (OBzl)-O- (CH2)15CH3(6e)
According to the method for embodiment 9, from 1.9g (3.3mmol) Boc-Gly-Glu (OBzl)-O- (CH2)15CH31.6g is obtained
(95.0%) title compound, is colourless powder.ESI-MS(m/e)：516.5[M+H]+；
Embodiment 26 prepares HClGly-Glu (OBzl)-O- (CH2)17CH3(6f)
According to the method for embodiment 9, from 2g (3.3mmol) Boc-Gly-Glu (OBzl)-O- (CH2)17CH31.73g is obtained
(96.2%) title compound, is colourless powder.ESI-MS(m/e)：544.5[M+H]+；
Embodiment 27 prepares Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)7CH3(7a)
According to the method for embodiment 3 by 1.28g (3.6mmol) Boc-His (Boc) and 1.21g (3mmol) HClGly-
Glu(OBzl)-O-(CH2)7CH31.26g (60%) title compound is obtained, is colourless powder.ESI-MS(m/e)：704[M+H
]+；
Embodiment 28 prepares Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)9CH3(7b)
According to the method for embodiment 3 by 1.28g (3.6mmol) Boc-His (Boc) and 1.29g (3mmol) HClGly-
Glu(OBzl)-O-(CH2)9CH31.45g (66.2%) title compound is obtained, is colourless powder.ESI-MS(m/e)：732[M+
H]+；
Embodiment 29 prepares Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)11CH3(7c)
According to the method for embodiment 3 by 1.28g (3.6mmol) Boc-His (Boc) and 1.38g (3mmol) HClGly-
Glu(OBzl)-O-(CH2)11CH31.41g (62.1%) title compound is obtained, is colourless powder.ESI-MS(m/e)：760[M
+H]+；
Embodiment 30 prepares Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)13CH3(7d)
According to the method for embodiment 3 by 1.28g (3.6mmol) Boc-His (Boc) and 1.46g (3mmol) HClGly-
Glu(OBzl)-O-(CH2)13CH31.4g (59.2%) title compound is obtained, is colourless powder.ESI-MS(m/e)：788[M+
H]+；
Embodiment 31 prepares Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)15CH3(7e)
According to the method for embodiment 3 by 1.28g (3.6mmol) Boc-His (Boc) and 1.55g (3mmol) HClGly-
Glu(OBzl)-O-(CH2)15CH31.4g (57.3%) title compound is obtained, is colourless powder.ESI-MS(m/e)：816[M+
H]+；
Embodiment 32 prepares Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)17CH3(7f)
According to the method for embodiment 3 by 1.28g (3.6mmol) Boc-His (Boc) and 1.63g (3mmol) HClGly-
Glu(OBzl)-O-(CH2)17CH31.46g (57.7%) title compound is obtained, is colourless powder.ESI-MS(m/e)：844[M
+H]+；
Embodiment 33 prepares 2HClHis-Gly-Glu (OBzl)-O- (CH2)7CH3(8a)
By 1.2g (1.71mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)7CH3It is dissolved in 40ml hydrogen chloride
Ethyl acetate solution (4M) solution in, be stirred at room temperature 4 hours, TLC (ethyl acetate: petroleum ether, 1: 2) show raw material point disappear
Lose, reactant mixture is concentrated under reduced pressure, and residue adds a small amount of ether to be concentrated under reduced pressure to remove hydrogen chloride gas repeatedly.It is finally plus few
Residue is ground to form 1.25g (97%) title compound by amount ether, is colourless powder, is directly used in next step reaction.
ESI-MS(m/e)：579[M+H]+；
Embodiment 34 prepares 2HClHis-Gly-Glu (OBzl)-O- (CH2)9CH3(8b)
According to the method for embodiment 33, from 1.4g (1.92mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)9CH31.05g (90.0%) title compound is obtained, is colourless powder.ESI-MS(m/e)：607[M+H]+；
Embodiment 35 prepares 2HClHis-Gly-Glu (OBzl)-O- (CH2)11CH3(8c)
According to the method for embodiment 33, from 1.4g (1.84mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O-
(CH2)11CH31.06g (90.9%) title compound is obtained, is colourless powder.ESI-MS(m/e)：635[M+H]+；
Embodiment 36 prepares 2HClHis-Gly-Glu (OBzl)-O- (CH2)13CH3(8d)
According to the method for embodiment 33, from 1.4g (1.78mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O-
(CH2)13CH31.12g (95%) title compound is obtained, is colourless powder.ESI-MS(m/e)：663[M+H]+；
Embodiment 37 prepares 2HClHis-Gly-Glu (OBzl)-O- (CH2)15CH3(8e)
According to the method for embodiment 33, from 1.4g (1.72mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O-
(CH2)15CH31.14g (96.4%) title compound is obtained, is colourless powder.ESI-MS(m/e)：691[M+H]+；
Embodiment 38 prepares 2HClHis-Gly-Glu (OBzl)-O- (CH2)17CH3(8f)
According to the method for embodiment 33, from 1.4g (1.66mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O-
(CH2)17CH31.12g (93.9%) title compound is obtained, is colourless powder.ESI-MS(m/e)：719[M+H]+；
Embodiment 39 prepares 2HClHis-Gly-Glu-O- (CH2)7CH3(9a)
By 1g (1.73mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)7CH3Dissolved with ethanol, add palladium
Carbon (Pd/C, reaction volume 10%), be passed through H2, it is stirred at room temperature to raw material point and disappears.Filter Pd/C, filtrate decompression concentration, system
0.88g (97%) title compound is obtained, is colourless powder.ESI-MS(m/e)：529[M+H]+；
Embodiment 40 prepares 2HClHis-Gly-Glu-O- (CH2)9CH3(9b)
According to the method for embodiment 39, from 1g (1.65mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)9CH30.88g (96.0%) title compound is obtained, is colourless powder.ESI-MS(m/e)：557[M+H]+；
Embodiment 41 prepares 2HClHis-Gly-Glu-O- (CH2)11CH3(9c)
According to the method for embodiment 39, from 1g (1.58mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)11CH30.89g (96.9%) title compound is obtained, is colourless powder.ESI-MS(m/e)：585[M+H]+；
Embodiment 42 prepares 2HClHis-Gly-Glu-O- (CH2)13CH3(9d)
According to the method for embodiment 39, from 1g (1.51mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)13CH30.91g (98.4%) title compound is obtained, is colourless powder.ESI-MS(m/e)：613[M+H]+；
Embodiment 43 prepares 2HClHis-Gly-Glu-O- (CH2)15CH3(9e)
According to the method for embodiment 39, from 1g (1.45mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)15CH30.89g (96.4%) title compound is obtained, is colourless powder.ESI-MS(m/e)：641[M+H]+；
Embodiment 44 prepares 2HClHis-Gly-Glu-O- (CH2)17CH3(9f)
According to the method for embodiment 39, from 1g (1.39mmol) Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)17CH30.87g (93.9%) title compound is obtained, is colourless powder.ESI-MS(m/e)：669[M+H]+；
Embodiment 45 prepares dexamethasone -3- formyl His-Gly-Glu-O- (CH2)7CH3Propionic ester (10a)
After 1.34g (2.28mmol) dexamethasone 3-N- formyl succinyl phosphorons amino propyl acids ester (2) are dissolved with 20ml DMF
Plus 0.8g (1.52mmol) 2HClHis-Gly-Glu-O- (CH2)7CH3, pH8 is adjusted with NMM, lucifuge is reacted 72 hours, TLC
(CH2Cl2∶CH3OH: HOAC, 5: 1: 0.15) show that raw material disappears substantially.Reaction solution is concentrated under reduced pressure into dry, residue 20ml first
Alcohol extracting 5 times, methanol solution is concentrated under reduced pressure, the yellow oil for obtaining 20ml ether: acetone (5: 1) solution foam washing 6 times, residual
(dichloromethane: methyl alcohol, 10: 1) obtains 0.51g (36.0%) title compound, is colourless powder to stay thing column chromatography to purify.
ESI-MS(m/e)：928[M+H]+；Mp167-170℃；IR(KBr)：3394,2935,
2360,1660,1573,1406,1203,1147,1051,893,673,516cm-1；1H NMR (300MHZ, DMSO-d6)：δ/
Ppm=9.86 (t, 1H), 9.21 (d, 1H), 8.32 (t, 1H), 7.46 (m, 1H), 7.34 (d, J=10.2Hz, 1H), 6.70 (m,
1H), 6.21 (d, J=10.2Hz, 1H), 6.00 (s, 1H), 5.39 (s, 1H), 5.15 (s, 1H), 5.05 (d, J=17.7Hz,
1H), 4.47 (d, 1H), 4.15 (m, 2H), 4.05 (m, 1H), 4.02 (m, 2H), 3.70 (m, 2H), 3.34 (m, 2H), 3.07 (m,
1H), 2.47 (m, 2H), 2.35 (m, 2H), 2.22 (m, 2H), 2.17 (m, 2H), 1.87 (m, 2H), 1.83 (m, 2H), 1.80 (m,
1H), 1.76 (m, 2H), 1.75 (m, 2H), 1.74 (m, 2H), 1.72 (m, 2H), 1.50 (s, 3H), 1.55-1.00 (m, 8H),
(d, J=6.9Hz, the 3H) of 1.34 (m, 2H), 1.3 (m, 1H), 1.08 (m, 1H), 0.89 (s, 3H), 0.83 (t, 3H), 0.79
Embodiment 46 prepares dexamethasone -3- formyl His-Gly-Glu-O- (CH2)9CH3Propionic ester (10b)
According to the method for embodiment 45, from 1.27g (2.16mmol) dexamethasone 3-N- formyl succinyl phosphorons amino propyl acids
Ester (2) and 0.8g (1.44mmol) 2HClHis-Gly-Glu (OBzl)-O- (CH2)9CH30.5g (36.0%) is obtained titled
Compound, is colourless powder.ESI-MS(m/e)：956[M+H]+；Mp173-175℃； IR
(KBr)：3410,2931,2360,1660,1593,1406,1398,1203,1070,893,675,607cm-1；1H NMR
(300MHZ, DMSO-d6)：δ/ppm=9.86 (t, 1H), 9.21 (d, 1H), 8.32 (t, 1H), 7.46 (m, 1H), 7.34 (d, J
=10.2Hz, 1H), 6.70 (m, 1H), 6.21 (d, J=10.2Hz, 1H), 6.00 (s, 1H), 5.39 (s, 1H), 5.15 (s,
1H), 5.05 (d, J=17.7Hz, 1H), 4.47 (d, 1H), 4.15 (m, 2H), 4.05 (m, 1H), 4.02 (m, 2H), 3.70 (m,
2H), 3.34 (m, 2H), 3.07 (m, 1H), 2.47 (m, 2H), 2.35 (m, 2H), 2.22 (m, 2H), 2.17 (m, 2H), 1.87 (m,
2H), 1.83 (m, 2H), 1.80 (m, 1H), 1.76 (m, 2H), 1.75 (m, 2H), 1.74 (m, 2H), 1.72 (m, 2H), 1.50 (s,
3H), 1.55-1.00 (m, 12H), 1.34 (m, 2H), 1.3 (m, 1H), 1.08 (m, 1H), 0.89 (s, 3H), 0.83 (t, 3H),
0.79 (d, J=6.9Hz, 3H)
Embodiment 47 prepares dexamethasone -3- formyl His-Gly-Glu-O- (CH2)11CH3Propionic ester (10c)
According to the method for embodiment 45, from 1.21g (2.06mmol) dexamethasone 3-N- formyl succinyl phosphorons amino propyl acids
Ester (2) and 0.8g (1.37mmol) 2HClHis-Gly-Glu (OBzl)-O- (CH2)11CH30.54g (40.0%) title is obtained
Compound, is colourless powder.ESI-MS(m/e)：984[M+H]+；Mp179-181℃；
IR(KBr)：3267,2935,2364,1662,1575,1421,1186,891,648,455cm-1；1H NMR (300MHZ,
DMSO-d6)：δ/ppm=9.86 (t, 1H), 9.21 (d, 1H), 8.32 (t, 1H), 7.46 (m, 1H), 7.34 (d, J=10.2Hz,
1H), 6.70 (m, 1H), 6.21 (d, J=10.2Hz, 1H), 6.00 (s, 1H), 5.39 (s, 1H), 5.15 (s, 1H), 5.05 (d, J
=17.7Hz, 1H), 4.47 (d, 1H), 4.15 (m, 2H), 4.05 (m, 1H), 4.02 (m, 2H), 3.70 (m, 2H), 3.34 (m,
2H), 3.07 (m, 1H), 2.47 (m, 2H), 2.35 (m, 2H), 2.22 (m, 2H), 2.17 (m, 2H), 1.87 (m, 2H), 1.83 (m,
2H), 1.80 (m, 1H), 1.76 (m, 2H), 1.75 (m, 2H), 1.74 (m, 2H), 1.72 (m, 2H), 1.50 (s, 3H), 1.55-
1.00 (m, 16H), 1.34 (m, 2H), 1.3 (m, 1H), 1.08 (m, 1H), 0.89 (s, 3H), 0.83 (t, 3H), 0.79 (d, J=
6.9Hz, 3H)
Embodiment 48 prepares dexamethasone -3- formyl His-Gly-Glu-O- (CH2)13CH3Propionic ester (10d)
According to the method for embodiment 45, from 1.16g (1.97mmol) dexamethasone 3-N- formyl succinyl phosphorons amino propyl acids
Ester (2) and 0.8g (1.31mmol) 2HClHis-Gly-Glu (OBzl)-O- (CH2)13CH30.51g (38.4%) title is obtained
Compound, is colourless powder.ESI-MS(m/e)：1012[M+H]+；Mp197-200℃；
IR(KBr)：3751,2978,2360,1664,1571,1423,1199,883,796,644cm-1；1H NMR (300MHZ,
DMSO-d6)：δ/ppm=9.86 (t, 1H), 9.21 (d, 1H), 8.32 (t, 1H), 7.46 (m, 1H), 7.34 (d, J=10.2Hz,
1H), 6.70 (m, 1H), 6.21 (d, J=10.2Hz, 1H), 6.00 (s, 1H), 5.39 (s, 1H), 5.15 (s, 1H), 5.05 (d, J
=17.7Hz, 1H), 4.47 (d, 1H), 4.15 (m, 2H), 4.05 (m, 1H), 4.02 (m, 2H), 3.70 (m, 2H), 3.34 (m,
2H), 3.07 (m, 1H), 2.47 (m, 2H), 2.35 (m, 2H), 2.22 (m, 2H), 2.17 (m, 2H), 1.87 (m, 2H), 1.83 (m,
2H), 1.80 (m, 1H), 1.76 (m, 2H), 1.75 (m, 2H), 1.74 (m, 2H), 1.72 (m, 2H), 1.50 (s, 3H), 1.55-
1.00 (m, 20H), 1.34 (m, 2H), 1.3 (m, 1H), 1.08 (m, 1H), 0.89 (s, 3H), 0.83 (t, 3H), 0.79 (d, J=
6.9Hz, 3H)
Embodiment 49 prepares dexamethasone -3- formyl His-Gly-Glu-O- (CH2)15CH3Propionic ester (10e)
According to the method for embodiment 45, from 1.10g (1.88mmol) dexamethasone 3-N- formyl succinyl phosphorons amino propyl acids
Ester (2) and 0.8g (1.25mmol) 2HClHis-Gly-Glu (OBzl)-O- (CH2)15CH30.47g (36.4%) title is obtained
Compound, is colourless powder.ESI-MS(m/e)：1040[M+H]+；Mp201-203℃；
IR(KBr)：3350,2927,2364,1664,1583,1404,1209,1037,808,677cm-1；1H NMR (300MHZ,
DMSO-d6)：δ/ppm=9.86 (t, 1H), 9.21 (d, 1H), 8.32 (t, 1H), 7.46 (m, 1H), 7.34 (d, J=10.2Hz,
1H), 6.70 (m, 1H), 6.21 (d, J=10.2Hz, 1H), 6.00 (s, 1H), 5.39 (s, 1H), 5.15 (s, 1H), 5.05 (d, J
=17.7Hz, 1H), 4.47 (d, 1H), 4.15 (m, 2H), 4.05 (m, 1H), 4.02 (m, 2H), 3.70 (m, 2H), 3.34 (m,
2H), 3.07 (m, 1H), 2.47 (m, 2H), 2.35 (m, 2H), 2.22 (m, 2H), 2.17 (m, 2H), 1.87 (m, 2H), 1.83 (m,
2H), 1.80 (m, 1H), 1.76 (m, 2H), 1.75 (m, 2H), 1.74 (m, 2H), 1.72 (m, 2H), 1.50 (s, 3H), 1.55-
1.00 (m, 24H), 1.34 (m, 2H), 1.3 (m, 1H), 1.08 (m, 1H), 0.89 (s, 3H), 0.83 (t, 3H), 0.79 (d, J=
6.9Hz, 3H) embodiments 50
Embodiment 50 prepares dexamethasone -3- formyl His-Gly-Glu-O- (CH2)17CH3Propionic ester (10f)
According to the method for embodiment 45, from 1.06g (1.80mmol) dexamethasone 3-N- formyl succinyl phosphorons amino propyl acids
Ester (2) and 0.8g (1.2mmol) 2HClHis-Gly-Glu (OBzl)-O- (CH2)17CH30.43g (33.9%) this reality is obtained
Apply the CH of example3(CH2)17OH compounds, are colourless powder.ESI-MS(m/e)：1068[M+H]+；Mp191-194℃；IR(KBr)：3296,2926,2360,1664,1575,1423,1182,802,651,
453cm-1；1H NMR (300MHZ, DMSO-d6)：δ/ppm=9.86 (t, 1H), 9.21 (d, 1H), 8.32 (t, 1H), 7.46
(m, 1H), 7.34 (d, J=10.2Hz, 1H), 6.70 (m, 1H), 6.21 (d, J=10.2Hz, 1H), 6.00 (s, 1H), 5.39
(s, 1H), 5.15 (s, 1H), 5.05 (d, J=17.7Hz, 1H), 4.47 (d, 1H), 4.15 (m, 2H), 4.05 (m, 1H), 4.02
(m, 2H), 3.70 (m, 2H), 3.34 (m, 2H), 3.07 (m, 1H), 2.47 (m, 2H), 2.35 (m, 2H), 2.22 (m, 2H), 2.17
(m, 2H), 1.87 (m, 2H), 1.83 (m, 2H), 1.80 (m, 1H), 1.76 (m, 2H), 1.75 (m, 2H), 1.74 (m, 2H), 1.72
(m, 2H), 1.50 (s, 3H), 1.55-1.00 (m, 28H), 1.34 (m, 2H), 1.3 (m, 1H), 1.08 (m, 1H), 0.89 (s,
3H), 0.83 (t, 3H), 0.79 (d, J=6.9Hz, 3H)
Experimental example 1 determines the nanometer particle size of 10a-f
10a-f is configured to 1 μM of the aqueous solution, in the upper 25 DEG C of surveys of laser nano particle size analyzer (Zetasizer, NANO-S90)
Determine particle diameter.METHOD FOR CONTINUOUS DETERMINATION 7 days, records its particle diameter.Result lists table 1 in.As shown by data, 10a-f nanoparticle warps in aqueous
Stabilization is in 150-420nm.
Nanometer particle size (nm) of the 1 10a-f aqueous solution of table in 7 days
Experimental example 2 determines the transmission electron microscope photo of 10a-f
It is 1 × 10 that 10a-f is made into concentration-6M, 1 × 10-9M, 1 × 10-11The aqueous solution of M, then drops in copper by this solution
On the net, nano shape is observed under JEM-1230 transmission electron microscopes after the dry solvent of volatilization.Measure shows that 10a-f forms rule
Nanosphere then.It is that concentration is 1 × 10 as representative graph 2-6The transmission electron microscope photo of the 10a-f of M.Result shows that 10a-f's receives
Rice structure is nanosphere of the diameter less than 200nm.Wherein ball of the diameter less than 100nm accounts for the overwhelming majority.It can be seen that, 10a-f's receives
Rice structure is very beneficial for them and conveys in vivo.
Experimental example 3 evaluates the inhibitory activity that 10a-f breeds to mouse spleen lymphocyte mitogen
De- neck puts to death mouse, aseptic to take spleen, is ground with 200 mesh steel meshes and piston, is washed twice with PBS liquid, 1500 turns/
Separate the heart 10 minutes, splenoblast 5 × 10 is matched somebody with somebody with complete RPMI-1640 nutrient solutions after counting6The cell suspension of/mL, plus 100 μ L
Cell suspension (contains 5 × 10 in 96 well culture plates per empty5Individual cell).Add 20 μ L (canavaline ConA, final concentration of 5 μ per hole
G/mL), 96 porocyte culture plates are placed in the CO that volume fraction is 0.05237 DEG C of culture 4h in the incubator of saturated humidity.4h
The RPMI-1640 nutrient solutions containing 0.5%DMSO of the 10a-f of sterilized treatment are added to match somebody with somebody solution (1 by default concentration gradient afterwards
×10-4, 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 are right
Isometric RPMI-1640 nutrient solutions containing 0.5%DMSO are added to match somebody with somebody according to group.Each Kong Jun is repeated 3 times (n=3).Culture 48h
Afterwards with mtt assay detection compound to the inhibitory action of SPL.
By inhibiting rate=[(OD that the OD average values -10a-f of the RPMI-1640 nutrient solution groups containing 0.5%DMSO is organized is average
Value)/containing 0.5%DMSO RPMI-1640 nutrient solution groups OD average values] × 100% calculate various concentrations 10a-f to spleen lymph
The inhibitory action of cell propagation, the concentration according to cell relative survival rate and 10a-f draws cell growth curve, using the growth
Curve try to achieve half inhibiting rate (DxIC50).This experimental data statistics is checked and variance analysis using t, with Represent.
Result lists table 2 in.Result shows to suppress the IC of mice spleen lymphocytes proliferation in 10a-f50It is 1.3-32 μM.Illustrate 10a-f formulas
Be conjugated and can keep the immunosuppressive activity of dexamethasone.
The inhibitory action of the 10a-f mouse spleen lymphocytes mitogen of table 2 propagation
N=3
Experimental example 4 evaluates the influence that 10a-f is transplanted to mouse ear rear myocardium tissue
Acceptor mouse (Balb/c mouse, male, 20 ± 2g of body weight) is through 10% urethane (10mg/10g body weight) intraperitoneal injection
Anesthesia.1% bromogeramine tincture auricle partly sterilised, hold eye scissors 1/3 before auricle dorsal part center line at make one with auricle center line
Vertical place makees a 3-4 millimeters of otch long, does not damage auricle vein.Tweezers are held to have sharp ears direction blunt separation hypodermis, is made
Into a tube chamber.New life is placed in trash ice into 75% alcohol skin after one minute for mouse (C57bl/6j24 hours suckling mouse) to disappear
Poison, cuts open chest and wins heart.Heart is placed in PBS liquid and beats 1-2 times to empty blood more than the chambers of the heart.During transplanting, the heart is supplied with blade handle
Longitudinal direction is cutd open as the big two halves such as substantially, and muscle fibre is into an inclined-plane.Cardiac muscular tissue's transplanting is inserted in acceptor mouse ear chamber, myocardium group
The isolated time knitted is no more than 2 minutes.With finger flicking part, graft is set to be adjacent to the surrounding tissue by mouse.Post-transplantation
The same day is administered.Blank be 0.5% sodium carboxymethylcellulose, 0.5% sodium carboxymethylcellulose of 10a-f and dexamethasone,
It is administered orally, dosage is 1.43 μm of ol/kg/d, 0.2mL/20g body weight, successive administration 15 days is administered 15 times altogether.
The electrocardiosignal of the cardiac muscular tissue of record transplanting daily from postoperative 7th day.During test dystopy electrocardiogram, positive and negative electrode
Heart transplant both sides are respectively placed in, earthing pole is connected to mouse hind leg.15 days after operation terminates observation, when statistics heart transplant is survived
Between.Data list table 3 in, and statistics is checked and variance analysis using t, withRepresent.Result shows, in 1.43 μ
10a-f significantly suppresses to transplant the rejection of cardiac muscular tissue under mol/kg/d dosage.The activity of wherein 10b, c, f is than ground plug rice
Song Qiang.
Influences of the table 310a-f to the mouse ear rear myocardium tissue time-to-live
N=10；A) with sodium carboxymethylcellulose than P ＜ 0.01；B) with sodium carboxymethylcellulose than P ＜ 0.01, and than ground
Sai meter Song is than P ＜ 0.05；C) with sodium carboxymethylcellulose and dexamethasone than P ＜ 0.01.
Experimental example 5 evaluates the influence that various dose 10c is transplanted to mouse ear rear myocardium tissue
In order to investigate the influence that dosage is transplanted to 10a-f to mouse ear rear myocardium tissue, selection 10c is representative, with experiment
The method of example 4, takes 1.43 μm of ol/kg/day, and 0.143 μm of ol/kg/day and 0.0143 μm of ol/kg/day Three doses is investigated
The influence that 10c is transplanted to mouse ear rear myocardium tissue.Data list table 4 in, and statistics is checked and variance analysis using t, withRepresent.Result shows that 10c dose-dependants ground suppresses the rejection of transplanting cardiac muscular tissue.It can be seen that, 10a-f pairs
The influence of mouse ear rear myocardium tissue transplanting has dose dependent.
Influences of the various dose 10c of table 4 to the mouse ear rear myocardium tissue time-to-live
N=10；A) the P ＜ 0.01 compared with sodium carboxymethylcellulose, with 0.143 μm of ol/kg/d10c than P ＜ 0.05；B) with
Sodium carboxymethylcellulose compares P ＜ 0.01, with 0.0143 μm of ol/kg/d10c than P ＜ 0.05；C) with sodium carboxymethylcellulose phase
Than P ＜ 0.05.
Experimental example 6 evaluates the analgesic activities of 10c in mouse heat radiation whipping model
In order to evaluate the analgesic activities of 10a-f, lived using 10c as the evaluation analgesia in mouse heat radiation whipping model is represented
Property.Dexamethasone and the equal single administrations of 10c, oral dose are 1.43 μm of ol/kg.Sodium carboxymethylcellulose is blank.
ICR male mices (20 ± 2g of body weight) test the previous day, and fasting can't help water, body weight is weighed before administration, averages, used as every
The dosage foundation of mouse, mouse is placed in self-control mouse cage, and afterbody is exposed, and mouse cage is fixed on table top, with 75%
Ethanol cleans rat-tail, is made marks at lower 1/3rd, as point of irradiation.Theral radiation instrument uses 220V, 35W, Qiang Shi
English bulb, light source forms light beam and is irradiated in survey pain position through outer cover optically focused funnel.When surveying pain, by funnel light source near survey pain portion
Position 5mm, radiation murine tail induced pain after focusing, with mouse TFL as pain reaction indicator, by manual time-keeping.First survey four
Secondary Basic Pain Threshold value, allows mouse that 30min is adapted in mouse cage before survey.30min after the oral administration of each group is measured,
Pain threshold when 60min, 90min, 120min, 150min and 180min, surveys three times every time, takes its average.Finally calculate the threshold of pain
Increase rate.Pain domain increase rate=[(pain thresholding after administration-basis pain thresholding)/Basic Pain Threshold value] × 100%. experimental datas systems
Meter is checked and variance analysis using t, withRepresent.Result lists table 5 in.
Result shows, under 1.43 μm of oL/kg, mouse can be significantly improved to hot spoke in administration 30min to 120min10c
Tolerance is penetrated, 60min to 90min analgesic activities reach peak value.With dexamethasone ratio, 10c is rapid-action, and 30min just works, and makees
It is long with the time, still there is obvious analgesic activity to 120min.
The influence that the 10c of table 5 is improved to mouse pain domain
N=10；A) the P ＜ 0.01 compared with sodium carboxymethylcellulose；B) P compared with sodium carboxymethylcellulose and dexamethasone
＜ 0.01；C) the P ＜ 0.05. compared with sodium carboxymethylcellulose and dexamethasone
Experimental example 7 evaluates the analgesic activities of various dose 10f in mouse heat radiation whipping model
In order to investigate influence of the dosage to 10a-f analgesic activities, selection 10c is representative, with the method for experimental example 6, is taken
1.43 μm of ol/kg, 0.51 μm of ol/kg and 0.25 μm of ol/kg Three doses investigate the analgesic activities of 10c.Data list table 6 in, system
Meter is checked and variance analysis using t, withRepresent.Result shows that the analgesic activities show dose of 10c is relied on
Property.It can be seen that, the analgesic activities of 10a-f have dose dependent.
The influence that the various dose 10c of table 6 is improved to mouse pain domain
N=10；A) the P ＜ 0.01 compared with sodium carboxymethylcellulose, the P ＜ 0.05 compared with 0.51 μm of ol/kg10c；B) with
Sodium carboxymethylcellulose and 0.25 μm of ol/kg10c compare P ＜ 0.01；C) the P ＜ 0.05 compared with sodium carboxymethylcellulose；D) with
Sodium carboxymethylcellulose is compared, P ＜ 0.01.
The anti-inflammatory activity of mouse caused by dimethylbenzene xylene inflammation model evaluation 10f of experimental example 8
In order to evaluate the anti-inflammatory activity of 10a-f, anti-inflammatory is evaluated on mouse caused by dimethylbenzene xylene inflammation model as representative using 10c
Activity.Dexamethasone and the equal single administrations of 10c, the oral dose of dexamethasone is 25.5 μm of ol/kg, and the oral dose of 10c is equal
It is 1.43 μm of oL/kg.Sodium carboxymethylcellulose is blank.ICR male mices (20 ± 2g of body weight) with and be divided into blank pair
According to group, positive group and administration group, every group 12.Tranquillization one day, it is 22 DEG C that operation room maintains temperature, and experiment starts, gastric infusion,
After single-dose 30min, the mortar that breaks toward the dimethylbenzene of the μ L of left auricle uniform application 30 of mouse, after 2h puts to death mouse, cuts a left side
Right two ears, circular auricle is taken with the card punch of 7mm in the same position of two ears, is weighed, and two ear swelling differences is obtained, as swelling
Degree.Swelling=left ear disk weight-auris dextra disk weight.This experimental data statistics is checked and variance analysis using t, withRepresent.Data list table 7 in.Result shows that 10c has outstanding anti-inflammatory activity under 1.43 μm of oL/kg, with 25.5 μ
The anti-inflammatory activity of mol/kg dexamethasone quite, i.e., with ground fill in when the dosage of 10c is the 1/17 of dexamethasone dosage by anti-inflammatory activity
Rice pine is quite.It can be seen that the anti-inflammatory activity of 10c is stronger than dexamethasone a lot.I.e. the anti-inflammatory activity of the anti-inflammatory activity of 10a-f is filled in than ground
Meter Song Qiang is a lot.
Inhibitory action of the 10c of table 7 to mice ear
N=10；A) the P ＜ 0.05 compared with sodium carboxymethylcellulose, the P ＞ 0.05. compared with 25.5 μm of ol/kg dexamethasone
The anti-inflammatory activity of mouse caused by dimethylbenzene xylene inflammation model evaluation various dose 10c of experimental example 9
In order to investigate influence of the dosage to 10a-f analgesic activities, selection 10c is representative, with the method for experimental example 8, is taken
25.5 μm of ol/kg, 1.43 μm of ol/kg and 0.51 μm of ol/kg Three doses investigate the analgesic activities of 10c.Data list table 8 in, system
Meter is checked and variance analysis using t, withRepresent.Result shows that the anti-inflammatory activity show dose of 10c is relied on
Property.It can be seen that, the anti-inflammatory activity of 10a-f has dose dependent.
Inhibitory action of the various dose 10c of table 8 to mice ear
N=10；A) the P ＜ 0.01 compared with sodium carboxymethylcellulose, the P ＜ 0.05 compared with 1.43 μm of ol/kg10c；B) with
Sodium carboxymethylcellulose and 0.51 μm of ol/kg10c compare P ＜ 0.05；C) the P ＜ 0.05. compared with sodium carboxymethylcellulose
Experimental example 10 evaluates influences of the 10c to mouse femur
Whether have to evaluate 10a-f and cause the side effect of osteoporosis, using 10c as representing commenting in experimental example 4
After valency terminates, 10c and sodium carboxymethylcellulose and Dexamethasone group press former administering mode and dosage continues administration 25 days, i.e.,
It is administered 40 days altogether, induces mouse osteoporosis.Afterwards, mouse orbit takes blood, stands 40 minutes, low-temperature and high-speed centrifuge
(SPD111V, Thermo) is centrifuged 20 minutes with 30000 revs/min, is taken its supernatant and is obtained mice serum sample, is tested with calcium and tried
Agent box (microplate method) and determination of inorganic phosphorus kit (the direct development process of peacock green) determine Determination of Calcium in Serum content and phosphorus content.Take small
Mouse left femur, rejects clean musculature, with chloroform: methyl alcohol (2: 1) soaks (every time 3 hours), degreasing twice.After degreasing
Femur be placed in 120 DEG C of baking ovens dry 6 hours, dry weight is claimed after cooling.The femur of dry weight will have been determined, 800 DEG C of muffle furnaces have been placed in
Middle calcining 8 hours, claims ash weight after cooling.Ash weight and the ratio (g/g) of dry weight are calculated, bone mineral content (BMC) is obtained.Bone ash is molten
In 5% aqueous hydrochloric acid solution (1: 9), using calcium test kit (microplate method) and determination of inorganic phosphorus kit, (peacock green is direct
Development process) determine bone phosphorus content and calcium content of bone.The measurement of bone density uses X-CT dual intensity bone density OCTs, Measurement portion
Three sections that 1-2mm below proximal femur is chosen in position measure, and average, and obtain bone density value.Result lists the He of table 9 in
Table 10.This experimental data statistics is checked and variance analysis using t, with Represent.
The as shown by data of table 9,10c treats the blood calcium of mouse, and serium inorganic phosphorus, bone calcium and bone phosphorus are all small higher than dexamethasone in treatment
Mouse, it is suitable with sodium carboxymethylcellulose treatment mouse.The as shown by data of table 10, the bone density of 10c treatment mouse is also above ground plug
The mouse of rice pine treatment, it is suitable with sodium carboxymethylcellulose treatment mouse.It can be seen that, different from dexamethasone, 10c treatments will not draw
Play mouse and osteoporosis occurs.That is 10a-f does not have the osteoporosis side effect of dexamethasone sample.
Calcium and phosphorus content in the blood and bone of the 10c of table 9 treatment mouse
N=10；A) the P ＜ 0.05. compared with sodium carboxymethylcellulose
The 10c of table 10 treats the femur dry weight of mouse, bone ash weight, bone mineral content and bone density
N=10；A) the P ＜ 0.05 compared with sodium carboxymethylcellulose；B) the P ＜ 0.01 compared with sodium carboxymethylcellulose.
Claims (6)
1. dexamethasone-the His-Gly- that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity that formula 10a-f is represented are modified
Glu conjugates, n=7 in formula, 9,11,13,15,17,
2. the dexamethasone that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity that the 10a-f of claim 1 is represented are modified-
The preparation method of His-Gly-Glu conjugates, the method is comprised the following steps：
(1) at (Boc)2L-Gly is converted into Boc-Gly in the presence of O and NaOH；
(2) at (Boc)2L-His is converted into Boc-His (Boc) in the presence of O and triethylamine；
(3) in dicyclohexyl carbonyl diimine (DCC), N- hydroxy benzo triazoles (HOBt), anhydrous tetrahydro furan (THF), N- first
By Boc-Glu (OBzl) and saturated fatty alcohol [CH in the presence of base morpholine (NMM)3(CH2) nOH] condensation, generation saturation Boc-Glu
(OBzl)-O-(CH2)nCH3；
(4) by Boc-Glu (OBzl)-O- (CH in the ethyl acetate solution (4M) of hydrogen chloride2)nCH3Removing Boc, generates Glu
(OBzl)-O-(CH2)nCH3；
(5) in DCC, HOBt, anhydrous THF, by Glu (OBzl)-O- (CH in the presence of NMM2)nCH3With Boc-Gly condensations, generation
Boc-Gly-Glu(OBzl)-O-(CH2)nCH3；
(6) by Boc-Gly-Glu (OBzl)-O- (CH in the presence of the ethyl acetate solution (4M) of hydrogen chloride2)nCH3Removing Boc,
Generation Gly-Glu (OBzl)-O- (CH2)nCH3；
(7) in DCC, HOBt, anhydrous THF, by Gly-Glu (OBzl)-O- (CH in the presence of NMM2)nCH3Contracted with Boc-His (Boc)
Close, generation Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH2)nCH3；
(8) by Boc-His (Boc)-Gly-Glu (OBzl)-O- (CH in the presence of the ethyl acetate solution (4M) of hydrogen chloride2)
nCH3Removing Boc, generation His-Gly-Glu (OBzl)-O- (CH2)nCH3；
(9) by His-Gly-Glu (OBzl)-O- (CH2)nCH3Hydrogenolysis, generation His-Gly-Glu-O- (CH2)nCH3；
(10) at DMAP (DMAP), dexamethasone and succinic anhydride are condensed in the presence of anhydrous THF, generation ground plug
Rice pine 3- carboxyl propionic esters；
(11) in 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (HClEDC), anhydrous dimethyl base formyl
, be condensed for dexamethasone 3- carboxyls propionic ester and N-hydroxy-succinamide (HOSu) in the presence of NMM by amine (DMF), generation ground plug
Rice pine 3-N- formyl succinyl phosphorons amino propyl acid esters；
(12) by dexamethasone 3-N- formyl succinyl phosphorons amino propyl acid esters and His-Gly-Glu- in the presence of dry DMF, NMM
O-(CH2)nCH3Condensation, generation dexamethasone 3- formyl-His-Gly-Glu-O- (CH2)nCH3Propionic ester.
3. the dexamethasone that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity that the 10a-f of claim 1 is represented are modified-
Application of the His-Gly-Glu conjugates in immunosuppressive drug is prepared.
4. the dexamethasone that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity that the 10a-f of claim 1 is represented are modified-
Application of the His-Gly-Glu conjugates in anti-inflammatory drug is prepared.
5. the dexamethasone that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity that the 10a-f of claim 1 is represented are modified-
Application of the His-Gly-Glu conjugates in analgesic is prepared.
6. the dexamethasone that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity that the 10a-f of claim 1 is represented are modified-
Purposes of the His-Gly-Glu conjugates in micro emulsion or liposome targeted drug is prepared.
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