CN104211762A - Conjugates of saturated aliphatic chain alcohol, dexamethasone, and His-Gly-Lys, preparation, nano structure, and applications thereof - Google Patents
Conjugates of saturated aliphatic chain alcohol, dexamethasone, and His-Gly-Lys, preparation, nano structure, and applications thereof Download PDFInfo
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- CN104211762A CN104211762A CN201310225686.XA CN201310225686A CN104211762A CN 104211762 A CN104211762 A CN 104211762A CN 201310225686 A CN201310225686 A CN 201310225686A CN 104211762 A CN104211762 A CN 104211762A
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Abstract
The invention relates to conjugates of saturated aliphatic chain alcohol, dexamethasone, and His-Gly-Lys, preparation, a nano structure, and applications thereof. The invention discloses 6 saturated aliphatic chain alcohol modified dexamethasone-His-Gly-Lys 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-His-Gly-Lys the conjugate that the 6 kinds of saturated aliphatic chain alcohols that the present invention relates to formula 10a-f representative are modified, n=7 in saturated aliphatic chain alcohol, 9,11,13,15,17, relate to their preparation method, relate to their nanostructure, relate to their immunosuppression, anti-inflammatory and analgesic activities, relate to their osteoporosis side effect.The present invention by 10a-f to the restraining effect of splenic lymphocyte mitogen proliferative response and to the survival time after myocardium transplantation after mouse ear, show that they have outstanding immunosuppressive action, 10a-f is to the ear swelling degree of mouse caused by dimethylbenzene xylene inflammation in research, show that they have outstanding anti-inflammatory action, 10a-f is on the impact of mouse thermal radiation whipping time in research, show that they have outstanding analgesic activity, 10a-f is on the impact of mouse femur in research, shows that they do not have osteoporosis side effect.Thus 10a-f is with a wide range of applications preparing in immunosuppressive drug.The invention belongs to biomedicine field.
Background technology
Diseased organ is substituted always by clinical yearning with normal organ.In transplantation before 20th century, except corneal graft, mostly end in failure.1945, the mechanism of repulsion was inquired in the medical biotechnology scholar Peter plum Davao of Britain via dermatoplastic experiment model, pointed out that transplantation is unsuccessfully the repulsion caused because of immunocyte.Within 1949, find adrenocortical hormone, used adrenocortical hormone that the success ratio of transplantation is improved greatly, thus started the beginning that immunosuppressor is applied to transplantation.Immunosuppressor, as the sequential use such as ciclosporin A and FK506, makes the survival time of transplant organ extend further.Because these medicines exist easily degraded, bioavailability is not high, and toxic side effect is strong, the shortcomings such as treatment target and scheme limitation, so research neotype immunosuppressant comes into one's own always.Contriver recognizes, glucocorticosteroid has strong immunosuppressive activity on the one hand, has osteoporosis side effect on the one hand.Contriver also recognizes, His-Gly-Lys tripeptides has suppressor activity peptide on the one hand, has the effect suppressing bone loss on the one hand.Contriver also recognizes, by glucocorticosteroid and His-Gly-Lys tripeptides covalent attachment, not only can improve immunosuppressive activity, and can eliminate the side effect of bone loss.Contriver recognizes further, and by glucocorticosteroid, His-Gly-Lys tripeptides and saturated aliphatic chain three covalent attachment, not only can strengthen the side effect of immunosuppressive activity and elimination bone loss, also can form nanostructure and be conducive to conveying in body.According to these understanding, contriver proposes glucocorticosteroid, His-Gly-Lys tripeptides and the covalently bound invention of saturated aliphatic chain three.Compared with immunosuppressor, glucocorticosteroid of the present invention, the covalently bound invention of His-Gly-Lys three.Compared with immunosuppressor, glucocorticosteroid of the present invention, His-Gly-Lys tripeptides and the covalently bound immunosuppression of saturated aliphatic chain three have 5 creativeness proposed: 1) strong immunosuppressive activity; 2) side effect of bone loss is not had; 3) form nanostructure and be conducive to conveying in body; 4) strong anti-inflammatory activity; 5) strong analgesic activities.
Summary of the invention
First technical problem to be solved by this invention is, the dexamethasone-His-Gly-Lys conjugate that 6 kinds that provide formula 10a-f the to represent saturated aliphatic chain alcohols with immunosuppressive activity are modified.N=7 in 10a; N=9 in 10b; N=11 in 10c; N=13 in 10d; N=15 in 10e; N=17 in 10f;
Second technical problem to be solved by this invention is, the preparation method of the dexamethasone-His-Gly-Lys conjugate providing 6 of formula 10a ~ f kinds of saturated aliphatic chain alcohols with immunosuppressive activity to modify, and the method comprises the following steps:
(1) at (Boc)
2l-Gly is converted into Boc-Gly under existing by O and NaOH;
(2) at (Boc)
2under O and triethylamine exist, L-His is converted into Boc-His (Boc);
(3) at dicyclohexyl carbonyl diimine (DCC), N-hydroxy benzo triazole (HOBt), anhydrous tetrahydro furan (THF), by Boc-Lys (Fmoc) and saturated fatty alcohol condensation under N-methylmorpholine (NMM) exists, generate Boc-Lys (Fmoc)-O-(CH
2) nCH
3;
(4) in the ethyl acetate solution (4M) of hydrogenchloride by Boc-Lys (Fmoc)-O-(CH
2) nCH
3remove Boc, generate Lys (Fmoc)-O-(CH
2) nCH
3;
(5) at DCC, HOBt, by Lys (Fmoc)-O-(CH under anhydrous THF, NMM exist
2) nCH
3with Boc-Gly condensation, generate Boc-Gly-Lys (Fmoc)-O-(CH
2) nCH
3;
(6) in the ethyl acetate solution (4M) of hydrogenchloride by Boc-Gly-Lys (Fmoc)-O-(CH
2) nCH
3remove Boc, generate Gly-Lys (Fmoc)-O-(CH
2) nCH
3;
(7) at DCC, HOBt, by Gly-Lys (Fmoc)-O-(CH under anhydrous THF, NMM exist
2) nCH
3with Boc-His (Boc) condensation, generate Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2) nCH
3;
(8) hydrogenchloride ethyl acetate solution (4M) exist under by Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2) nCH
3remove Boc, generate His-Gly-Lys (Fmoc)-O-(CH
2) nCH
3;
(9) at DMAP (DMAP), by dexamethasone and Succinic anhydried condensation under anhydrous THF exists, dexamethasone 3-carboxyl propionic ester is generated;
(10) in 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (HClEDC), anhydrous dimethyl formamide (DMF), by dexamethasone 3-carboxyl propionic ester and N-hydroxy-succinamide (HOSu) condensation under NMM exists, generate dexamethasone 3-N-formyl succinyl phosphorons amino propyl acid ester;
(11) in dry DMF, by dexamethasone 3-N-formyl succinyl phosphorons amino propyl acid ester and His-Gly-Lys (Fmoc)-O-(CH under NMM exists
2) nCH
3condensation, generates dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
ncH
3propionic ester.
(12) at piperidines, under the existence of methylene dichloride, by dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
ncH
3propionic ester removes Fmoc, generates dexamethasone 3-formyl-His-Gly-Lys-O-(CH
2)
ncH
3propionic ester, to obtain final product.
The 3rd technical problem that the present invention solves studies by experiment, evaluates the nanometer character of the dexamethasone-His-Gly-Lys conjugate that 6 kinds of saturated aliphatic chain alcohols are modified;
The present invention solve the 4th technical problem be study by experiment, have rated dexamethasone-His-Gly-Lys conjugate that 6 kinds of saturated aliphatic chain alcohols modify to the restraining effect of mouse spleen lymphocyte to the proliferative response of mitogen;
The present invention solve the 5th technical problem be study by experiment, evaluates dexamethasone-His-Gly-Lys conjugate mouse ear rear myocardium tissue's transplanting survival time of 6 kinds of saturated aliphatic chain alcohols modifications;
The present invention solve the 6th technical problem be study by experiment, evaluates the dexamethasone-His-Gly-Lys conjugate mouse thermal radiation whipping reaction times of 6 kinds of saturated aliphatic chain alcohols modifications;
The present invention solve the 7th technical problem be study by experiment, evaluates the dexamethasone-His-Gly-Lys conjugate mouse caused by dimethylbenzene xylene inflammation ear swelling degree that 6 kinds of saturated aliphatic chain alcohols are modified;
The present invention solve the 8th technical problem be study by experiment, evaluates the dexamethasone-His-Gly-Lys conjugate of 6 kinds of saturated aliphatic chain alcohols modifications to the impact of mouse femur.
The present invention passes through the dexamethasone-His-Gly-Lys conjugate of the saturated aliphatic chain alcohol modification described in research to the restraining effect of splenic lymphocyte mitogen proliferative response, on the impact of the survival time after myocardium transplantation after mouse ear, indicate compound of the present invention and there is outstanding immunosuppressive action.Be with a wide range of applications preparing in immune suppressant drug.
One of purposes of the dexamethasone-His-Gly-Lys conjugate that saturated aliphatic chain alcohol of the present invention is modified has without the purposes in the steroidal immunosuppressor of osteoporosis effect in preparation;
Two of the purposes of the dexamethasone-His-Gly-Lys conjugate that saturated aliphatic chain alcohol of the present invention is modified is the purposes in the immunosuppressor preparing possess both anti-inflammatory analgesic activities.
Dexamethasone-His-Gly-Lys the conjugate that saturated aliphatic chain alcohol of the present invention is modified is preparing the purposes in micro emulsion or liposome targeted drug.
Accompanying drawing explanation
Fig. 1 is the synthetic route of the dexamethasone-His-Gly-Lys conjugate that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity of formula 10a-f representative are modified.I) anhydrous THF, DMAP; Ii) dry DMF, HClEDC; Iii) anhydrous THF, DMAP, HOBt and NMM; Iv) ethyl acetate solution (4M) of hydrogenchloride; V) dry DMF, NMM; Vi) 20% piperidines/dichloromethane solution; N=17 in n=15,10f in n=13,10e in n=11,10d in n=9,10c in n=7,10b in 10a.
Fig. 2 is that 10a-f is 1 × 10
-6the representative transmission electron microscope photo of the nanometer ball formed in the M aqueous solution.
Embodiment
In order to set forth the present invention further, provide a series of embodiment below.These embodiments are illustrative completely, and they are only used for being specifically described the present invention, not should be understood to limitation of the present invention.
Embodiment 1 prepares dexamethasone 3-carboxyl propionic ester (1)
3.93g (10mmol) dexamethasone reacts 48 hours, TLC (CH with 1.3g (13mmol) Succinic anhydried in the middle lucifuge of 150ml tetrahydrofuran (THF) (THF) under 1.46g (12mmol) DMAP (DM AP) exists
2cl
2: CH
3oH: HOAC, 20: 1: 0.15) raw material completely dissolve is shown.Reaction mixture adds 30ml H
2o, concentrating under reduced pressure, residue adds KHSO
4regulate pH2, the colourless powder acetone/sherwood oil of precipitation 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 acid ester (2)
4.92g (10mmol) dexamethasone succinate (1) reacts 24 hours, TLC (CH with 1.50g (13mmol) N-hydroxy-succinamide (HOSu) lucifuge in the solvent of 100ml THF and 10ml DMF under 2.50g (13mmol) 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (HClEDC) exists
2cl
2: CH
3oH: HOAC, 30: 1: 0.15) raw material completely dissolve is shown.Reaction mixture concentrating under reduced pressure, residue adds 150ml ethyl acetate, the saturated NaHCO of solution 20ml obtained
3the aqueous solution washes 3 times, washes 2 times with the saturated NaCl aqueous solution of 20ml, with the saturated KHSO of 20ml
4the aqueous solution washes 3 times, then 2 times are washed with the saturated NaCl aqueous solution of 20ml, ethyl acetate layer anhydrous sodium sulfate drying, filter, filtrate reduced in volume is extremely dry, residue column chromatography (sherwood oil: acetone, 1: 2) purifying, obtaining 5.37g (91%) title compound, is colourless powder.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-Lys (Fmoc)-O-(CH
2)
7cH
3(3a)
2.34g (5mmol) Boc-Lys (Fmoc) is dissolved in the anhydrous THF of 20mL, in the solution obtained, adds 0.675g (5mmol) N-hydroxy benzo triazole (HO Bt) under ice bath, and make it dissolve completely.Add 1.236g (6mmol) dicyclohexyl carbonyl diimine (DCC) after 10 minutes and obtain reaction solution I.An ice bath lower 0.78g (6mmol) CH
3(CH
2)
7oH is suspended in 20mL anhydrous methylene chloride, then adds 1mLN-methylmorpholine (NMM) and adjusts pH9.Stir 35 minutes, obtain reaction solution II.Ice bath lower reaction solution I adds in reaction solution II, stirs 1h under first ice bath, then stirring at room temperature 12h, TLC (ethyl acetate: sherwood oil, 2: 1) show Boc-Lys (Fmoc) disappearance.Filtering dicyclohexylurea (DCU) (DCU), concentrating under reduced pressure, residue 50mL acetic acid ethyl dissolution.The solution obtained uses saturated NaHCO successively
3the aqueous solution is washed, and the saturated NaCl aqueous solution is washed, 5%KHSO
4the aqueous solution is washed and is washed with the saturated NaCl aqueous solution.Ethyl acetate uses anhydrous Na mutually
2sO
4drying, filters, and filtrate reduced in volume is to dry, and obtaining 2.5g (85%) title compound, is colourless powder.ESI-MS(m/e)：581.54[M+H]
+；
Embodiment 4 prepares Boc-Lys (Fmoc)-O-(CH
2)
9cH
3(3b)
According to the preparation method of embodiment 3 by 2.34g (5mmol) Boc-Lys (Fmoc) and 0.948g (6mmol) CH
3(CH
2)
9oH obtains 2.6g (86%) title compound, is colourless powder.ESI-MS(m/e)：609.54[M+H]
+；
Embodiment 5 prepares Boc-Lys (Fmoc)-O-(CH
2)
11cH
3(3c)
According to the method for embodiment 3 by 2.34g (5mmol) Boc-Lys (Fmoc) and 1.116g (6mmol) CH
3(CH
2)
11oH obtains 3.0g (88.4%) title compound, is colourless powder.ESI-MS(m/e)：637.54[M+H]
+；
Embodiment 6 prepares Boc-Lys (Fmoc)-O-(CH
2)
13cH
3(3d)
According to the method for embodiment 3 by 2.34g (5mmol) Boc-Lys (Fmoc) and 1.284g (6mmol) CH
3(CH
2)
13oH obtains 3.1g (92.9%) title compound, is colourless powder.ESI-MS(m/e)：665.54[M+H]
+；
Embodiment 7 prepares Boc-Lys (Fmoc)-O-(CH
2)
15cH
3(3e)
According to the method for embodiment 3 by 2.34g (5mmol) Boc-Lys (Fmoc) and 1.452g (6mmol) CH
3(CH
2)
15oH obtains 2.9g (84.75%) title compound, is colourless powder.ESI-MS(m/e)：693.54[M+H]
+；
Embodiment 8 prepares Boc-Lys (Fmoc)-O-(CH
2)
17cH
3(3f)
According to the method for embodiment 3 by 2.34g (5mmol) Boc-Lys (Fmoc) and 1.62g (6mmol) CH
3(CH
2)
17oH obtains 2.9g (81.5%) title compound, is colourless powder.ESI-MS(m/e)：721.54[M+H]
+；
Embodiment 9 prepares HClLys (Fmoc)-O-(CH
2)
7cH
3(4a)
By 2.50g (4.3mmol) Boc-Lys (Fmoc)-O-(CH
2)
7cH
3be dissolved in ethyl acetate solution (4M) solution of 50ml hydrogenchloride, stirring at room temperature 2 hours, TLC (ethyl acetate: sherwood oil, 1: 2) shows raw material point and disappears, concentrating under reduced pressure, 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 and residue is ground to form 2.12g (95%) title compound, is colourless powder, is directly used in next step reaction.ESI-MS(m/e)：519.05[M+H]
+；
Embodiment 10 prepares HClLys (Fmoc)-O-(CH
2)
9cH
3(4b)
According to the method for embodiment 9, from 2.5g (4.1mmol) Boc-Lys (Fmoc)-O (CH
2)
9cH
3obtained 2.13g (95%) title compound is colourless powder.ESI-MS(m/e)：547.04[M+H]
+；
Embodiment 11 prepares HClLys (Fmoc)-O-(CH
2)
11cH
3(4c)
According to the method for embodiment 9, from 2.8g (4.4mmol) Boc-Lys (Fmoc)-O-(CH
2)
11cH
3obtained 2.33g (92.1%) title compound is colourless powder.ESI-MS(m/e)：575.04[M+H]
+；
Embodiment 12 prepares HClLys (Fmoc)-O-(CH
2)
13cH
3(4d)
According to the method for embodiment 9, from 2.8g (4.21mmol) Boc-Lys (Fmoc)-O-(CH
2)
13cH
3obtained 2.3g (89.2%) title compound is colourless powder.ESI-MS(m/e)：603.04[M+H]
+；
Embodiment 13 prepares HClLys (Fmoc)-O-(CH
2)
15cH
3(4e)
According to the method for embodiment 9, from 2.8g (4mmol) Boc-Lys (Fmoc)-O-(CH
2)
15cH
3obtained 2.4g (96.7%) title compound is colourless powder.ESI-MS(m/e)：631.04[M+H]
+；
Embodiment 14 prepares HClLys (Fmoc)-O-(CH
2)
17cH
3(4f)
According to the method for embodiment 9, from 2.8g (3.89mmol) Boc-Lys (Fmoc)-O-(CH
2)
17cH
3obtained 2.5g (95%) title compound is colourless powder.ESI-MS(m/e)：659.04[M+H]
+；
Embodiment 15 prepares Boc-Gly-Lys (Fmoc)-O-(CH
2)
7cH
3(5a)
According to the method for embodiment 3 by 0.8g (4.56mmol) Boc-Gly and 2.0g (3.86mmol) HClLys (Fmoc)-O-(CH
2)
7cH
3obtained 2.3g (92.2%) title compound is colourless powder.ESI-MS(m/e)：638[M+H]
+；
Embodiment 16 prepares Boc-Gly-Lys (Fmoc)-O-(CH
2)
9cH
3(5b)
According to the method for embodiment 3 by 0.8g (4.56mmol) Boc-Gly and 2.1g (3.86mmol) HClLys (Fmoc)-O-(CH
2)
9cH
3obtained 2.35g (91.7%) title compound is colourless powder.ESI-MS(m/e)：666[M+H]
+；
Embodiment 17 prepares Boc-Gly-Lys (Fmoc)-O-(CH
2)
11cH
3(5c)
According to the method for embodiment 3 by 0.8g (4.56mmol) Boc-Gly and 2.2g (3.86mmol) HClLys (Fmoc)-O-(CH
2)
11cH
3obtained 2.34g (87.8%) title compound is colourless powder.ESI-MS(m/e)：694[M+H]
+；
Embodiment 18 prepares Boc-Gly-Lys (Fmoc)-O-(CH
2)
13cH
3(5d)
According to the method for embodiment 3 by 0.8g (4.56mmol) Boc-Gly and 2.3g (3.86mmol) HClLys (Fmoc)-O-(CH
2)
13cH
3obtained 2.7g (98%) title compound is colourless powder.ESI-MS(m/e)：722[M+H]
+；
Embodiment 19 prepares Boc-Gly-Lys (Fmoc)-O-(CH
2)
15cH
3(5e)
According to the method for embodiment 3 by 0.8g (4.56mmol) Boc-Gly and 2.4g (3.86mmol) HClLys (Fmoc)-O (CH
2)
15cH
3obtained 2.6g (92%) title compound is colourless powder.ESI-MS(m/e)：750[M+H]
+；
Embodiment 20 prepares Boc-Gly-Lys (Fmoc)-O-(CH
2)
17cH
3(5f)
According to the method for embodiment 3 by 0.8g (4.56mmol) Boc-Gly and 2.5g (3.86mmol) HClLys (Fmoc)-O-(CH
2)
17cH
3obtained 2.8g (93.4%) title compound is colourless powder.ESI-MS(m/e)：778[M+H]
+；
Embodiment 21 prepares HClGly-Lys (Fmoc)-O-(CH
2)
7cH
3(6a)
According to the method for embodiment 9, from 2.0g (3.1mmol) Boc-Gly-Lys (Fmoc)-O-(CH
2)
7cH
3obtained 1.7g (95.4%) title compound is colourless powder.ESI-MS(m/e)：575[M+H]
+；
Embodiment 22 prepares HClGly-Lys (Fmoc)-O-(CH
2)
9cH
3(6b)
According to the method for embodiment 9, from 2.0g (3.0mol) Boc-Gly-Lys (Fmoc)-O-(CH
2)
9cH
3obtained 1.74g (96%) title compound is colourless powder.ESI-MS(m/e)：603[M+H]
+；
Embodiment 23 prepares HClGly-Lys (Fmoc)-O-(CH
2)
11cH
3(6c)
According to the method for embodiment 9, from 2.0g (2.9mmol) Boc-Gly-Lys (Fmoc)-O-(CH
2)
11cH
3obtained 1.7g (93.5%) title compound is colourless powder.ESI-MS(m/e)：631[M+H]
+；
Embodiment 24 prepares HClGly-Lys (Fmoc)-O-(CH
2)
13cH
3(6d)
According to the method for embodiment 9, from 2.2g (3.1mmol) Boc-Gly-Lys (Fmoc)-O-(CH
2)
13cH
3obtained 2.0g (99.6%) title compound is colourless powder.ESI-MS(m/e)：659[M+H]
+；
Embodiment 25 prepares HClGly-Lys (Fmoc)-O-(CH
2)
15cH
3(6e)
According to the method for embodiment 9, from 2.3g (3.1mmol) Boc-Gly-Lys (Fmoc)-O-(CH
2)
15cH
3obtained 2.0g (95.0%) title compound is colourless powder.ESI-MS(m/e)：687[M+H]
+；
Embodiment 26 prepares HClGly-Lys (Fmoc)-O-(CH
2)
17cH
3(6f)
According to the method for embodiment 9, from 2.5g (3.2mmol) Boc-Gly-Lys (Fmoc)-O-(CH
2)
17cH
3obtained 2.2g (96.2%) title compound is colourless powder.ESI-MS(m/e)：715[M+H]
+；
Embodiment 27 prepares Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
7cH
3(7a)
According to the method for embodiment 3 by 1.19g (3.34mmol) Boc-His (Boc) and 1.6g (2.78mmol) HClGly-Lys (Fmoc)-O-(CH
2)
7cH
3obtained 1.58g (65%) title compound is pale yellow oil.ESI-MS(m/e)：875[M+H]
+；
Embodiment 28 prepares Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
9cH
3(7b)
According to the method for embodiment 3 by 1.13g (3.19mmol) Boc-His (Boc) and 1.6g (2.66mmol) HClGly-Lys (Fmoc)-O-(CH
2)
9cH
3obtained 1.56g (65%) title compound is pale yellow oil.ESI-MS(m/e)：903[M+H]
+；
Embodiment 29 prepares Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
11cH
3(7c)
According to the method for embodiment 3 by 1.08g (3.04mmol) Boc-His (Boc) and 1.6g (2.53mmol) HClGly-Lys (Fmoc)-O-(CH
2)
11cH
3obtained 1.58g (67%) title compound is colorless oil.ESI-MS(m/e)：931[M+H]
+；
Embodiment 30 prepares Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
13cH
3(7d)
According to the method for embodiment 3 by 1.16g (3.28mmol) Boc-His (Boc) and 1.8g (2.73mmol) HClGly-Lys (Fmoc)-O-(CH
2)
13cH
3obtained 1.65g (63%) title compound is colorless oil.ESI-MS(m/e)：959[M+H]
+；
Embodiment 31 prepares Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
15cH
3(7e)
According to the method for embodiment 3 by 1.12g (3.14mmol) Boc-His (Boc) and 1.8g (2.62mmol) HClGly-Lys (Fmoc)-O-(CH
2)
15cH
3obtained 1.55g (60%) title compound is colorless oil.ESI-MS(m/e)：987[M+H]
+；
Embodiment 32 prepares Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
17cH
3(7f)
According to the method for embodiment 3 by 1.19g (3.36mmol) Boc-His (Boc) and 2.0g (2.8mmol) HClGly-Lys (Fmoc)-O-(CH
2)
17cH
3obtained 1.53g (54%) title compound is colorless oil.ESI-MS(m/e)：1015[M+H]
+；
Embodiment 33 prepares 2HClHis-Gly-Lys (Fmoc)-O-(CH
2)
7cH
3(8a)
By 1.5g (1.71mmol) Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
7cH
3be dissolved in ethyl acetate solution (4M) solution of 40ml hydrogenchloride, stirring at room temperature 4 hours, TLC (ethyl acetate: sherwood oil, 1: 2) show raw material point to disappear, reaction mixture concentrating under reduced pressure, 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 and residue is ground to form 1.25g (97%) title compound, is colourless powder, is directly used in next step reaction.ESI-MS(m/e)：750[M+H]
+；
Embodiment 34 prepares 2HClHis-Gly-Lys (Fmoc)-O-(CH
2)
9cH
3(8b)
According to the method for embodiment 33, from 1.5g (1.66mmol) Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
9cH
3obtained 1.26g (98%) title compound is pale yellow powder.ESI-MS(m/e)：778[M+H]
+；
Embodiment 35 prepares 2HClHis-Gly-Lys (Fmoc)-O-(CH
2)
11cH
3(8c)
According to the method for embodiment 33, from 1.5g (1.61mmol)
Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
11cH
3obtained 1.25g (95%) title compound is pale yellow powder.ESI-MS(m/e)：806[M+H]
+；
Embodiment 36 prepares 2HClHis-Gly-Lys (Fmoc)-O-(CH
2)
13cH
3(8d)
According to the method for embodiment 33, from 1.5g (1.56mmol) Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
13cH
3obtained 1.25g (96%) title compound is pale yellow powder.ESI-MS(m/e)：834[M+H]
+；
Embodiment 37 prepares 2HClHis-Gly-Lys (Fmoc)-O-(CH
2)
15cH
3(8e)
According to the method for embodiment 33, from 1.5g (1.52mmol) Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
15cH
3obtained 1.27g (97%) title compound is pale yellow powder.ESI-MS(m/e)：862[M+H]
+；
Embodiment 38 prepares 2HClHis-Gly-Lys (Fmoc)-O-(CH
2)
17cH
3(8f)
According to the method for embodiment 33, from 1.5g (1.48mmol) Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2)
17cH
3obtained 1.25g (95%) title compound is pale yellow powder.ESI-MS(m/e)：890[M+H]
+；
Embodiment 39 prepares dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
7cH
3propionic ester (9a)
1.18g (2mmol) dexamethasone 3-N-formyl succinyl phosphorons amino propyl acid ester (2) adds 1g (1.34mmol) 2HClHis-Gly-Lys (Fmoc)-O-(CH after dissolving with 20ml DMF
2)
7cH
3, regulate pH8 with NMM, lucifuge reacts 72 hours, TLC (CH
2cl
2: CH
3oH: HOAC, 5: 1: 0.15) show raw material to disappear.Reaction solution is evaporated to dry, residue 20ml methanol extraction 5 times, methanol solution concentrating under reduced pressure, the yellow oil 20ml ether obtained: acetone (5: 1) solution foam washing 6 times, residue column chromatography purification (methylene dichloride: methyl alcohol, 15: 1) obtaining 1.23g (80%) title compound, is colorless oil.ESI-MS(m/e)：1151[M+H]
+。
Embodiment 40 prepares dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
9cH
3propionic ester (9b)
According to the method for embodiment 39, from 1.14g (1.94mmol) dexamethasone 3-N-formyl succinyl phosphorons amino propyl acid ester (2) and 1g (1.29mmol) 2HClHis-Gly-Lys (Fmoc)-O-(CH
2)
9cH
3obtained 1.21g (80%) title compound is colorless oil.ESI-MS(m/e)：1180[M+H]
+。
Embodiment 41 prepares dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
11cH
3propionic ester (9c)
According to the method for embodiment 39, from 1.09g (1.86mmol) dexamethasone 3-N-formyl succinyl phosphorons amino propyl acid ester (2) and 1g (1.24mmol) 2HClHis-Gly-Lys (Fmoc)-O-(CH
2)
11cH
3obtained 1.16g (78%) title compound is colorless oil.ESI-MS(m/e)：1208[M+H]
+。
Embodiment 42 prepares dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
13cH
3propionic ester (9d)
According to the method for embodiment 39, from 1.06g (1.8mmol) dexamethasone 3-N-formyl succinyl phosphorons amino propyl acid ester (2) and 1g (1.2mmol) 2HClHis-Gly-Lys (Fmoc)-O-(CH
2)
13cH
3obtained 1.16g (78%) title compound is colorless oil.ESI-MS(m/e)：1233[M+H]
+。
Embodiment 43 prepares dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
15cH
3propionic ester (9e)
According to the method for embodiment 39, from 1.06g (1.8mmol) dexamethasone 3-N-formyl succinyl phosphorons amino propyl acid ester (2) and 1g (1.2mmol) 2HClHis-Gly-Glu (OBzl)-O-(CH
2)
15cH
3obtained 1.3g (86%) title compound is colorless oil.ESI-MS(m/e)：1261[M+H]
+。
Embodiment 44 prepares dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
17cH
3propionic ester (9f)
According to the method for embodiment 39, from 1.06g (1.8mmol) dexamethasone 3-N-formyl succinyl phosphorons amino propyl acid ester (2) and 1g (1.1mmol) 2HClHis-Gly-Glu (OBzl)-O-(CH
2)
17cH
3obtained 1.2g (86%) title compound is colorless oil.ESI-MS(m/e)：1289[M+H]
+。
Embodiment 45 prepares dexamethasone 3-formyl-His-Gly-Lys-O-(CH
2)
7cH
3propionic ester (10a)
By 1g (0.9mmol) dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
7cH
3propionic ester is dissolved in 10ml20% piperidines/dichloromethane solution, lucifuge reaction 30min.Reaction mixture is evaporated to dry, and residue 100ml ether solidification, filters, and obtained 0.7g (95%) title compound is colourless powder.ESI-MS(m/e)：927[M+H]
+。Mp139-143℃。
IR(KBr)：3371，2941，2357，1660，1546，1452，1392，1263，1093，806，671cm
-1。
1H?NMR(300MHZ，DMSO-d6)：δ/ppm＝8.50(t，1H)，8.43(d，J＝7.2Hz，1H)，8.31(t，1H)，8.17(d，J＝6.6Hz，1H)，7.87(d，J＝6.6Hz，1H)，7.50(m，1H)，7.34(d，J＝10.2Hz，1H)，6.75(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)，4.78(d，J＝17.7Hz，1H)，4.50(m，1H)，4.16(m，2H)，4.02(t，2H)，3.80(m，1H)，3.54(m，2H)，3.07(m，1H)，2.87(m，2H)，2.85(m，2H)，2.72(m，2H)，2.57(m，2H)，2.32(m，2H)，2.16(m，2H)，1.90(m，1H)，1.76(m，2H)，1.70(m，2H)，1.66(m，2H)，1.60(m，2H)，1.55-1.00(m，10H)，1.49(s，3H)，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 46 prepares dexamethasone 3-formyl-His-Gly-Lys-O-(CH
2)
9cH
3propionic ester (10b)
According to the method for embodiment 45, from 1g (0.9mmol) dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
9cH
3propionic ester obtains 0.77g (95%) title compound, is colourless powder.ESI-MS(m/e)：955[M+H]
+。Mp139-143℃。
IR(KBr)：3394，2939，2349，1660，1539，1448，1396，1197，1058，806，677cm
-1。
1H?NMR(300MHZ，DMSO-d6)：δ/ppm＝8.50(t，1H)，8.43(d，J＝7.2Hz，1H)，8.31(t，1H)，8.17(d，J＝6.6Hz，1H)，7.87(d，J＝6.6Hz，1H)，7.50(m，1H)，7.34(d，J＝10.2Hz，1H)，6.75(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)，4.78(d，J＝17.7Hz，1H)，4.50(m，1H)，4.16(m，2H)，4.02(t，2H)，3.80(m，1H)，3.54(m，2H)，3.07(m，1H)，2.87(m，2H)，2.85(m，2H)，2.72(m，2H)，2.57(m，2H)，2.32(m，2H)，2.16(m，2H)，1.90(m，1H)，1.76(m，2H)，1.70(m，2H)，1.66(m，2H)，1.60(m，2H)，1.55-1.00(m，14H)，1.49(s，3H)，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-Lys-O-(CH
2)
11cH
3propionic ester (10c)
According to the method for embodiment 45, from 1g (0.8mmol) dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
11cH
3propionic ester obtains 0.7g (91%) title compound, is colourless powder.ESI-MS(m/e)：983[M+H]
+。Mp139-143℃。
IR(KBr)：3294，2939，2351，1660，1541，1448，1396，1209，1024，769，677cm
-1。
1H?NMR(300MHZ，DMSO-d6)：δ/ppm＝8.50(t，1H)，8.43(d，J＝7.2Hz，1H)，8.31(t，1H)，8.17(d，J＝6.6Hz，1H)，7.87(d，J＝6.6Hz，1H)，7.50(m，1H)，7.34(d，J＝10.2Hz，1H)，6.75(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)，4.78(d，J＝17.7Hz，1H)，4.50(m，1H)，4.16(m，2H)，4.02(t，2H)，3.80(m，1H)，3.54(m，2H)，3.07(m，1H)，2.87(m，2H)，2.85(m，2H)，2.72(m，2H)，2.57(m，2H)，2.32(m，2H)，2.16(m，2H)，1.90(m，1H)，1.76(m，2H)，1.70(m，2H)，1.66(m，2H)，1.60(m，2H)，1.55-1.00(m，18H)，1.49(s，3H)，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-Lys-O-(CH
2)
13cH
3propionic ester (10d)
According to the method for embodiment 45, from 1g (0.8mmol) dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
13cH
3propionic ester obtains 0.75g (93%) title compound, is colourless powder.ESI-MS(m/e)：1011[M+H]
+。Mp139-143℃。
IR(KBr)：3375，2943，2347，1666，1541，1450，1406，1219，1031，923，663cm
-1。
1H?NMR(300MHZ，DMSO-d6)：δ/ppm＝8.50(t，1H)，8.43(d，J＝7.2Hz，1H)，8.31(t，1H)，8.17(d，J＝6.6Hz，1H)，7.87(d，J＝6.6Hz，1H)，7.50(m，?1H)，7.34(d，J＝10.2Hz，1H)，6.75(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)，4.78(d，J＝17.7Hz，1H)，4.50(m，1H)，4.16(m，2H)，4.02(t，2H)，3.80(m，1H)，3.54(m，2H)，3.07(m，1H)，2.87(m，2H)，2.85(m，2H)，2.72(m，2H)，2.57(m，2H)，2.32(m，2H)，2.16(m，2H)，1.90(m，1H)，1.76(m，2H)，1.70(m，2H)，1.66(m，2H)，1.60(m，2H)，1.55-1.00(m，22H)，1.49(s，3H)，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-Lys-O-(CH
2)
15cH
3propionic ester (10e)
According to the method for embodiment 45, from 1g (0.8mmol) dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
15cH
3propionic ester obtains 0.7g (92%) title compound, is colourless powder.ESI-MS(m/e)：1039[M+H]
+。Mp139-143℃。
IR(KBr)：3275，2933，2351，1660，1535，1448，1394，1193，1062，889，682cm
-1。
1H?NMR(300MHZ，DMSO-d6)：δ/ppm＝8.50(t，1H)，8.43(d，J＝7.2Hz，1H)，8.31(t，1H)，8.17(d，J＝6.6Hz，1H)，7.87(d，J＝6.6Hz，1H)，7.50(m，1H)，7.34(d，J＝10.2Hz，1H)，6.75(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)，4.78(d，J＝17.7Hz，1H)，4.50(m，1H)，4.16(m，2H)，4.02(t，2H)，3.80(m，1H)，3.54(m，2H)，3.07(m，1H)，2.87(m，2H)，2.85(m，2H)，2.72(m，2H)，2.57(m，2H)，2.32(m，2H)，2.16(m，2H)，1.90(m，1H)，1.76(m，2H)，1.70(m，2H)，1.66(m，2H)，1.60(m，2H)，1.55-1.00(m，26H)，1.49(s，3H)，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 50 prepares dexamethasone 3-formyl-His-Gly-Lys-O-(CH
2)
17cH
3propionic ester (10f)
According to the method for embodiment 45, from 1g (0.78mmol) dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
17cH
3propionic ester obtains 0.79g (95%) title compound, is colourless powder.ESI-MS(m/e)：1067[M+H]
+。Mp139-143℃。
IR(KBr)：3298，2937，2345，1666，1535，1438，1398，1182，663，547，441cm
-1。
1H?NMR(300MHZ，DMSO-d6)：δ/ppm＝8.50(t，1H)，8.43(d，J＝7.2Hz，1H)，8.31(t，1H)，8.17(d，J＝6.6Hz，1H)，7.87(d，J＝6.6Hz，1H)，7.50(m，1H)，7.34(d，J＝10.2Hz，1H)，6.75(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)，4.78(d，J＝17.7Hz，1H)，4.50(m，1H)，4.16(m，2H)，4.02(t，2H)，3.80(m，1H)，3.54(m，2H)，3.07(m，1H)，2.87(m，2H)，2.85(m，2H)，2.72(m，2H)，2.57(m，2H)，2.32(m，2H)，2.16(m，2H)，1.90(m，1H)，1.76(m，2H)，1.70(m，2H)，1.66(m，2H)，1.60(m，2H)，1.55-1.00(m，30H)，1.49(s，3H)，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 measures the nanometer particle size of 10a-f
10a-f is configured to the aqueous solution of 1 μM, measures particle diameter upper 25 DEG C of laser nano particle size analyzer (Zetasizer, NANO-S90).METHOD FOR CONTINUOUS DETERMINATION 7 days, records its particle diameter.Result lists table 1 in.Data show, 10a-f nanoparticle is in aqueous through being stabilized in 150-350nm.
Nanometer particle size (nm) in the table 1 10a-f aqueous solution 7 days
Experimental example 2 measures the transmission electron microscope photo of 10a-f
10a-f being made into concentration is 1 × 10
-6m, 1 × 10
-9m, 1 × 10
-11the aqueous solution of M, then drops on copper mesh by this solution, observes nano shape after the dry solvent that volatilizees under JEM-1230 transmission electron microscope.Mensuration shows, the nanometer ball of 10a-f formation rule.Representatively Fig. 2 is concentration is 1 × 10
-6the transmission electron microscope photo of the 10a-f of M.Result shows, the nanostructure of 10a-f is the nanometer ball that diameter is less than 200nm.The ball that wherein diameter is less than 100nm accounts for the overwhelming majority.Visible, the nanostructure of 10a-f is very beneficial for them and carries in vivo.
Experimental example 3 evaluates the inhibit activities that 10a-f breeds mouse spleen lymphocyte mitogen
De-neck puts to death mouse, asepticly gets spleen, and grind with 200 order steel meshes and piston, wash twice with PBS liquid, 1500 revs/min centrifugal 10 minutes, is made into splenocyte 5 × 10 after counting with complete RPMI-1640 nutrient solution
6the cell suspension of/mL, adds 100 μ L cell suspensions (every empty containing 5 × 10 in 96 well culture plates
5individual cell).Every hole adds 20 μ L (canavaline ConA, final concentration is 5 μ g/mL), and this 96 porocyte culture plate is placed in the CO that volume fraction is 0.05
24h is cultivated for 37 DEG C in the incubator of saturated humidity.The RPMI-1640 nutrient solution containing 0.5%DMSO added through the 10a-f of sterilising treatment by the concentration gradient preset after 4h joins solution (1 × 10
-4, 8 × 10
-5, 5 × 10
-5, 2 × 10
-5, 1 × 10
-5, 8 × 10
-6, 5 × 10
-6, 1 × 10
-6), the multiple hole of each concentration 3, control group adds isopyknic RPMI-1640 nutrient solution containing 0.5%DMSO and joins.Each hole is all repeated 3 times (n=3).Use mtt assay detection compound to the restraining effect of splenic lymphocyte after cultivating 48h.
Different concns 10a-f is calculated to the restraining effect of spleen lymphocyte proliferation by inhibiting rate=[(the OD mean value that the OD mean value-10a-f containing the RPMI-1640 nutrient solution group of 0.5%DMSO organizes)/containing the OD mean value of the RPMI-1640 nutrient solution group of 0.5%DMSO] × 100%, concentration according to cell relative survival rate and 10a-f draws cell growth curve, utilize this growth curve try to achieve half inhibiting rate (
dxiC
50).This experimental data statistics all adopts t inspection and variance analysis, with
represent.Result lists table 2 in.Result shows the IC suppressing mice spleen lymphocytes proliferation at 10a-f
50for 3-71 μM.What 10a-f formula was described puts together the immunosuppressive activity that can keep dexamethasone.
The restraining effect of table 2 10a-f mouse spleen lymphocyte mitogen propagation
n＝3
Experimental example 4 evaluates the impact that 10a-f transplants mouse ear rear myocardium tissue
Acceptor mouse (Balb/c mouse, male, body weight 20 ± 2g) through 10% urethane (10mg/10g body weight) intraperitoneal injection of anesthesia.1% bromogeramine tincture auricle partly sterilised, holds eye scissors 1/3 place's work one and auricle median perpendicular place before auricle dorsal part center line and makes the otch of a 3-4 millimeters long, do 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 trash ice 75% alcohol skin degerming after a minute for mouse (C57bl/6j24 hour suckling mouse), cuts open chest and win heart.Heart is placed in PBS liquid and beats 1-2 time with blood more than the emptying chambers of the heart.During transplanting, become the large two halves such as basic with blade longitudinally cuing open for the heart, myofiber becomes an inclined-plane.Cardiac muscular tissue being transplanted inserts in acceptor mouse ear chamber, and the isolated time of cardiac muscular tissue is no more than 2 minutes.With finger flicking local, graft is adjacent to the surrounding tissue by mouse.Post-transplantation administration on the same day.Blank is 0.5% Xylo-Mucine, 10a-f and dexamethasone 0.5% Xylo-Mucine, equal oral administration, and dosage is 1.43 μm of ol/kg/day, 0.2mL/20g body weight, successive administration 15 days, altogether administration 15 times.
Within postoperative 7th day, play the electrocardiosignal recording heart transplantation muscular tissue every day.During test ectocardia electrograph, positive and negative electrode is placed in heart transplant both sides respectively, and earthing pole is connected to mouse hind leg.15 days after operation terminates to observe, statistics prolonged Survival of Rat Cardiac.Data list table 3 in, and statistics all adopts t inspection and variance analysis, with
represent.Result shows, under 1.43 μm of ol/kg/day dosage, 10a-f suppresses the rejection of heart transplantation muscular tissue significantly.Wherein the specific activity dexamethasone of 10a, d, e, f is strong.
Table 3 10a-f is on the impact of mouse ear rear myocardium tissue survival time
N=10; A) with Xylo-Mucine than P < 0.01; B) with Xylo-Mucine than P < 0.01, with than dexamethasone than P < 0.05; C) with Xylo-Mucine and dexamethasone than P < 0.01.
Experimental example 5 evaluates the impact that various dose 10f transplants mouse ear rear myocardium tissue
In order to investigate the impact that dosage is transplanted mouse ear rear myocardium tissue 10a-f, selection 10f is representative, by the method for experimental example 4, get 1.43 μm of ol/kg/day, the impact that 0.143 μm of ol/kg/day and 0.0143 μm of ol/kg/day Three doses investigation 10f transplants mouse ear rear myocardium tissue.Data list table 4 in, and statistics all adopts t inspection and variance analysis, with
represent.Result shows, 10f dose-dependant ground suppresses the rejection of heart transplantation muscular tissue.Visible, 10a-f has dose-dependently to the impact that mouse ear rear myocardium tissue transplants.
Table 4 various dose 10f is on the impact of mouse ear rear myocardium tissue survival time
N=10; A) P < 0.01 compared with Xylo-Mucine, with 0.143 μm of ol/kg/d10f than P < 0.05; B) P < 0.01 compared with Xylo-Mucine, with 0.0143 μm of ol/kg/d10f than P < 0.05; C) compared with Xylo-Mucine, P < 0.05.
Experimental example 6 evaluates the analgesic activities of 10f on mouse thermal radiation whipping model
In order to evaluate the analgesic activities of 10a-f, representatively on mouse thermal radiation whipping model, evaluate analgesic activities with 10f.Dexamethasone and the equal single administration of 10f, oral dosage is 1.43 μm of ol/kg.Xylo-Mucine is blank.ICR male mice (body weight 20 ± 2g) tests the day before yesterday, water is can't help in fasting, take body weight before administration, average, as the dosage foundation of every mouse, mouse is placed in self-control mouse cage, afterbody is exposed, and mouse tail, on table top, is cleaned with 75% ethanol by fixing mouse cage, make marks, as point of irradiation in lower 1/3rd places.Theral radiation instrument adopts 220V, 35W, strong quartz bulb, and light source forms light beam irradiation in survey pain position through outer cover optically focused funnel.When surveying pain, by funnel light source near surveying pain position 5mm, radiation murine tail induced pain after focusing on, with mouse TFL for pain indicator reaction, passes through manual time-keeping.First survey four Basic Pain Threshold values, before survey, allow mouse adapt to 30min in mouse cage.Measure threshold of pain when 30min, 60min, 90min, 120min, 150min and 180min after oral administration of each group, each to survey three times, get its average.Finally calculate threshold of pain increase rate.Pain territory increase rate=[(after administration pain thresholding-basis pain thresholding)/Basic Pain Threshold value] × 100%. experimental data statistics all adopt t inspection and variance analysis, with
represent.Result lists table 5 in.
Result shows, under 1.43 μm of oL/kg, can significantly improve mouse to thermal radiation tolerance at administration 30min to 120min 10f, 60min to 90min analgesic activity reaches peak value.With dexamethasone ratio, 10f is rapid-action, and 30min is with regard to onset, and long action time, still has obvious analgesic activity to 120min.
The impact that table 5 10f improves mouse pain territory
N=10; A) P < 0.01 compared with Xylo-Mucine; B) P < 0.01 compared with Xylo-Mucine and dexamethasone; C) P < 0.05. compared with Xylo-Mucine and dexamethasone
Experimental example 7 evaluates the analgesic activities of various dose 10f on mouse thermal radiation whipping model
In order to investigate the impact of dosage on 10a-f analgesic activities, selection 10f is representative, by the method for experimental example 6, gets 1.43 μm of ol/kg, and 0.51 μm of ol/kg and 0.25 μm of ol/kg Three doses investigates the analgesic activities of 10f.Data list table 6 in, and statistics all adopts t inspection and variance analysis, with
represent.Result shows, the analgesic activities show dose dependency of 10f.Visible, the analgesic activities of 10a-f has dose-dependently.
The impact that table 6 various dose 10f improves mouse pain territory
N=10; A) P < 0.01 compared with Xylo-Mucine and 0.51 μm of ol/kg10f; B) P < 0.01 compared with Xylo-Mucine, P < 0.05 compared with 0.51 μm of ol/kg10f; C) P < 0.01 compared with Xylo-Mucine and 0.25 μm of ol/kg10f; D) P < 0.01 compared with Xylo-Mucine, P < 0.05 compared with 0.25 μm of ol/kg10f; E) P < 0.05 compared with Xylo-Mucine, P < 0.01 compared with 0.25 μm of ol/kg10f; F) P < 0.05 compared with Xylo-Mucine; G) compared with Xylo-Mucine, P < 0.01.
Experimental example 8 anti-inflammatory activity of the scorching model evaluation 10f of mouse caused by dimethylbenzene xylene
In order to evaluate the anti-inflammatory activity of 10a-f, representatively on the scorching model of mouse caused by dimethylbenzene xylene, evaluate anti-inflammatory activity with 10f.Dexamethasone and the equal single administration of 10f, the oral dosage of dexamethasone is that the oral dosage of 25.5 μm of ol/kg, 10f is 1.43 μm of oL/kg.Xylo-Mucine is blank.ICR male mice (body weight 20 ± 2g) with and be divided into blank group, positive group and administration group, often organize 12.Tranquillization one day, operation room holding temperature is 22 DEG C, and experiment starts, gastric infusion, after single-dose 30min, toward the dimethylbenzene of the left auricle uniform application 30 μ L of mouse, after 2h, mouse put to death by disconnected mortar, cuts left and right two ears, circular auricle is got in the same position of two ears with the punch tool of 7mm, weigh, obtain two ear swelling differences, as swelling.Swelling=left ear disk weight-auris dextra disk weight.This experimental data statistics all adopts t inspection and variance analysis, with
represent.Data list table 7 in.Result shows, under 1.43 μm of oL/kg, 10f has outstanding anti-inflammatory activity, suitable with the anti-inflammatory activity of 25.5 μm of ol/kg dexamethasone, and when namely the dosage of 10f is 1/17 of dexamethasone dosage, anti-inflammatory activity is suitable with dexamethasone.The anti-inflammatory activity of visible 10f is much stronger than dexamethasone.Namely the anti-inflammatory activity of the anti-inflammatory activity of 10a-f is much stronger than dexamethasone.
Table 7 10f is to the restraining effect of mice ear
N=10; A) P < 0.05 compared with Xylo-Mucine, P > 0.05. compared with 25.5 μm of ol/kg dexamethasone
Experimental example 9 anti-inflammatory activity of the scorching model evaluation various dose 10f of mouse caused by dimethylbenzene xylene
In order to investigate the impact of dosage on 10a-f analgesic activities, selection 10f is representative, by the method for experimental example 7, gets 25.5 μm of ol/kg, and 1.43 μm of ol/kg and 0.51 μm of ol/kg Three doses investigate the analgesic activities of 10f.Data list table 8 in, and statistics all adopts t inspection and variance analysis, with
represent.Result shows, the anti-inflammatory activity show dose dependency of 10f.Visible, the anti-inflammatory activity of 10a-f has dose-dependently.
Table 8 various dose 10f is to the restraining effect of mice ear
N=10; A) P < 0.01 compared with Xylo-Mucine, P < 0.05 compared with 1.43 μm of ol/kg10f; B) P < 0.05 compared with Xylo-Mucine and 0.51 μm of ol/kg10f; C) P < 0.05. compared with Xylo-Mucine
Experimental example 10 evaluates the impact of 10f on mouse femur
Whether have cause osteoporotic side effect to evaluate 10a-f, with 10f representatively after the evaluation of experimental example 4 terminates, 10f and Xylo-Mucine and Dexamethasone group continue administration 25 days by former administering mode and dosage, and namely administration 40 days altogether, brings out mouse osteoporosis.Afterwards, mouse orbit gets blood, leave standstill 40 minutes, low-temperature and high-speed whizzer (SPD111V, Thermo) centrifugal 20 minutes with 30000 revs/min, get its supernatant and obtain mice serum sample, measure Determination of Calcium in Serum content and phosphorus content with calcium test kit (microplate method) and determination of inorganic phosphorus test kit (the direct development process of Victoria Green WPB).Get mouse left femur, reject clean muscle tissue, with chloroform: methyl alcohol (2: 1) soaks twice (each 3 hours), degreasing.Femur after degreasing is placed in 120 DEG C of baking ovens to dry 6 hours, after cooling, claims dry weight.To measure the femur of dry weight, be placed in 800 DEG C of muffle furnace calcinings 8 hours, after cooling, claim ash heavy.Calculate ash and weigh the ratio (g/g) with dry weight, obtain bone mineral content (BMC).Bone ash is dissolved in the aqueous hydrochloric acid (1: 9) of 5%, uses calcium test kit (microplate method) and determination of inorganic phosphorus test kit (the direct development process of Victoria Green WPB) to measure bone phosphorus content and calcium content of bone.The measurement of bone density adopts X-CT dual intensity bone density OCT, and three cross sections that below proximal femur 1-2mm is chosen in measuring point are measured, and average, obtain bone density value.Result lists table 9 and table 10 in.This experimental data statistics all adopts t inspection and variance analysis, with
represent.
The data of table 9 show, 10f treats the blood calcium of mouse, serium inorganic phosphorus, and bone calcium and bone phosphorus are all higher than the mouse of dexamethasone in treatment, and to treat mouse suitable with Xylo-Mucine.The data of table 10 show, 10f treats the bone density of mouse also higher than the mouse of dexamethasone in treatment, and to treat mouse suitable with Xylo-Mucine.Visible, different from dexamethasone, 10f treatment can not cause mouse generation osteoporosis.Namely 10a-f does not have the osteoporosis side effect of dexamethasone sample.
Table 9 10f treats calcium and phosphorus content in the blood of mouse and bone
N=10; A) P < 0.05. compared with Xylo-Mucine
Table 10 10f treats the femur dry weight of mouse, bone ash weight, bone mineral content and bone density
N=10; A) P < 0.05 compared with Xylo-Mucine; B) P < 0.01 compared with Xylo-Mucine.
Claims (9)
1. 6 kinds of formula 10a-f representative have the dexamethasone-His-Gly-Lys conjugate of the saturated aliphatic chain alcohol modification of immunosuppressive activity, n=7 in formula, 9,11,13,15,17.
。
2. the preparation method of the dexamethasone-His-Gly-Lys conjugate that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity that the 10a-f of claim 1 represents are modified, the method comprises the following steps:
(1) at (Boc)
2l-Gly is converted into Boc-Gly under existing by O and NaOH;
(2) at (Boc)
2under O and triethylamine exist, L-His is converted into Boc-His (Boc);
(3) at dicyclohexyl carbonyl diimine (DCC), N-hydroxy benzo triazole (HOBt), anhydrous tetrahydro furan (THF), by Boc-Lys (Fmoc) and saturated fatty alcohol condensation under N-methylmorpholine (NMM) exists, generate Boc-Lys (Fmoc)-O-(CH
2) nCH
3;
(4) in the ethyl acetate solution (4M) of hydrogenchloride by Boc-Lys (Fmoc)-O-(CH
2) nCH
3remove Boc, generate Lys (Fmoc)-O-(CH
2) nCH
3;
(5) at DCC, HOBt, by Lys (Fmoc)-O-(CH under anhydrous THF, NMM exist
2) nCH
3with Boc-Gly condensation, generate Boc-Gly-Lys (Fmoc)-O-(CH
2) nCH
3;
(6) in the ethyl acetate solution (4M) of hydrogenchloride by Boc-Gly-Lys (Fmoc)-O-(CH
2) nCH
3remove Boc, generate Gly-Lys (Fmoc)-O-(CH
2) nCH
3;
(7) at DCC, HOBt, by Gly-Lys (Fmoc)-O-(CH under anhydrous TH F, NMM exist
2) nCH
3with Boc-His (Boc) condensation, generate Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2) nCH
3;
(8) hydrogenchloride ethyl acetate solution (4M) exist under by Boc-His (Boc)-Gly-Lys (Fmoc)-O-(CH
2) nCH
3remove Boc, generate His-Gly-Lys (Fmoc)-O-(CH
2) nCH
3;
(9) at DMAP (DMAP), by dexamethasone and Succinic anhydried condensation under anhydrous THF exists, dexamethasone 3-carboxyl propionic ester is generated;
(10) in 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (HClEDC), anhydrous dimethyl formamide (DMF), by dexamethasone 3-carboxyl propionic ester and N-hydroxy-succinamide (HOSu) condensation under NMM exists, generate dexamethasone 3-N-formyl succinyl phosphorons amino propyl acid ester;
(11) in dry DMF, by dexamethasone 3-N-formyl succinyl phosphorons amino propyl acid ester and His-Gly-Lys (Fmoc)-O-(CH under NMM exists
2) nCH
3condensation, generates dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
ncH
3propionic ester.
(12) at piperidines, under the existence of methylene dichloride, by dexamethasone 3-formyl-His-Gly-Lys (Fmoc)-O-(CH
2)
ncH
3propionic ester removes Fmoc, generates dexamethasone 3-formyl-His-Gly-Lys-O-(CH
2)
ncH
3propionic ester, to obtain final product.
3. the nanostructure of the dexamethasone-His-Gly-Lys conjugate that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity that the 10a-f of claim 1 represents are modified.
4. the immunosuppressive activity of the dexamethasone-His-Gly-Lys conjugate that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity that the 10a-f of claim 1 represents are modified.
5. the anti-inflammatory activity of the dexamethasone-His-Gly-Lys conjugate that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity that the 10a-f of claim 1 represents are modified.
6. the analgesic activities of the dexamethasone-His-Gly-Lys conjugate that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity that the 10a-f of claim 1 represents are modified.
7. dexamethasone-the His-Gly-Lys that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity of the 10a-f representative of claim 1 are modified puts together the osteoporosis side effect of thing's vanished.
8. dexamethasone-His-Gly-Lys the conjugate that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity of the 10a-f representative of claim 1 are modified is preparing the application in immunosuppressive drug.
9. dexamethasone-His-Gly-Lys the conjugate that 6 kinds of saturated aliphatic chain alcohols with immunosuppressive activity of the 10a-f representative of claim 1 are modified is preparing the purposes in micro emulsion or liposome targeted drug.
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| 徐艳霞: "药物设计中的伪肽先导结构的发现", 《首都医科大学学报》 * |
| 王超: "基于甾体和多肽相互作用的化学和疗效", 《全国化学生物学学术会议》 * |
| 王超: "甾体-多肽缀合激素的合成及免疫抑制活性", 《药学学报》 * |
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| US10780174B2 (en) | 2015-12-23 | 2020-09-22 | The University Of British Columbia | Lipid-linked prodrugs |
| CN109381708A (en) * | 2018-07-18 | 2019-02-26 | 温州医科大学 | A kind of dexamethasone-polypeptide nano particle eye drops and preparation method thereof |
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