CN102796168A - Compounds with thrombolytic activity and preparation method and application thereof - Google Patents

Compounds with thrombolytic activity and preparation method and application thereof Download PDF

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CN102796168A
CN102796168A CN2011101411267A CN201110141126A CN102796168A CN 102796168 A CN102796168 A CN 102796168A CN 2011101411267 A CN2011101411267 A CN 2011101411267A CN 201110141126 A CN201110141126 A CN 201110141126A CN 102796168 A CN102796168 A CN 102796168A
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boc
och
ala
pro
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CN102796168B (en
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彭师奇
赵明
李国春
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Capital Medical University
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Capital Medical University
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Abstract

The invention provides 18 thrombolytic compounds (4a-r) and a preparation method thereof. The general formula I of the compounds is Pro-Ala-AA-OCH2(CH2)12CH3, wherein AA is a glycine residue, an L-valine residue, an L-tryptophan residue, an L-leucine residue, an L-alanine residue, an L-methionine residue, an L-tyrosine residue, an L-asparagine residue, an L-isoleucine residue, an L-phenylalanine residue, an L-proline residue, an L-serine residue, an L-threonine residue, an L-glutamine residue, an L-aspartic acid residue, an L-glutamic acid residue, an L-arginine residue or an L-lysine residue. The compounds have a thrombolytic effect on a rat carotid artery and jugular vein bypass cannula thrombus model and self-assembly performance. The 18 thrombolytic compounds provided by the invention have a good application prospect in clinical application.

Description

A kind of compound with thrombus dissolving activity,
Technical field
The present invention relates to a kind of compound of synthetic, particularly a kind of compound with thrombus dissolving activity also relates to its preparation method and the application in the preparation thrombolytic agent, the invention belongs to biomedicine field.
Background technology
At present; Treating the acute thrombus embolism class diseases clinically mainly is through using medicines such as streptokinase, urokinase and rt-PA to carry out this treatment of diseases; But adverse consequencess such as ubiquity systemic bleeding tendency and immunogenic response when using these medicines; Therefore, seeking a kind of safe and effective medicine is one of new drug research focus of treatment acute thrombus embolism class diseases.
P6A (ARPAK) is one of scleroproein β chain degradation product, has thrombus dissolving activity.In the metabolism research of P6A, found meta-bolites PAK.On rat arteriovenous shut intubate thrombus dissolving model, discover that the thrombus dissolving activity of PAK is stronger than parent P6A.According to general understanding, polypeptide all can be degraded rapidly in vivo.Structural modification through PAK and then delay polypeptide degradation rate in vivo and improve thrombus dissolving activity is one of important channel of thrombolytic agent research.
The amphipathic molecule that contains polypeptide, for example self-assembly can take place through intermolecular non-covalent interaction in aliphatic amide chain modified polypeptides under suitable condition, forms nanostructure.By nanostructure can improve in vivo conveying of polypeptide, delay polypeptide in vivo degradation rate and improve the activity in vivo of polypeptide.According to these understanding, the contriver has proposed the present invention.
Summary of the invention
One of the object of the invention provides the compound (representing with 4a-r) of the general formula I with thrombus dissolving activity:.
Pro-Ala-AA-OCH 2(CH 2) 12CH 3General formula I
In the formula, AA be selected from glycocoll (Gly) residue, L-Xie Ansuan (Val) residue, L-tryptophan residue (Trp), L-leucine residue (Leu), L-L-Ala (Ala) residue, L-methionine(Met) (Met) residue, L-tyrosine (Tyr) residue, altheine (Asn) residue, L-Isoleucine (Ile) residue, L-phenylalanine(Phe) (Phe) residue, L-proline(Pro) (Pro) residue, L-Serine (Ser) residue, L-Threonine (Thr) residue, L-glutaminate (Gln) residue, L-aspartic acid (Asp) residue, L-L-glutamic acid (Glu) residue, L-l-arginine (Arg) residue or L-Methionin (Lys) residue one of them;
4a is the compound of AA=glycine residue interval scale; 4b is the compound of AA=L-alanine residue interval scale; 4c is the compound of AA=L-phenylalanine residue interval scale; 4d is the compound of AA=L-Xie Ansuan residue interval scale; 4e is the compound of AA=L-leucine residue interval scale; 4f is the compound of AA=L-Isoleucine residue interval scale; 4g is the compound of AA=L-proline residue interval scale; 4h is the compound of AA=L-tyrosine residues interval scale; 4i is the compound of AA=L-tryptophan residue interval scale; 4j is the compound of AA=L-asparagicacid residue interval scale; 4k is the compound of AA=L-glutaminic acid residue interval scale; 4l is the compound of AA=L-arginine residues interval scale; 4m is the compound of AA=L-serine residue interval scale; 4n is the compound of AA=L-threonyl amine residue interval scale; 4o is the compound of AA=L-lysine residue interval scale; 4p is the compound of AA=L-methionine residues interval scale; 4q is the compound of AA=L-asparagine residue interval scale; 4r is the compound of AA=L-glutamine residue interval scale.
Two of the object of the invention provides a kind of above-mentioned method with thrombus dissolving activity compound for preparing, and it is characterized in that may further comprise the steps:
1) Boc-Pro is Boc-Pro-Ala with the Ala condensation in anhydrous THF in the presence of NSC 57182 (DCC) and N-hydroxy-succinamide (HOSu);
2) Boc-AA is Boc-AA-OCH with the condensation of carbon tetradecyl alcohol in anhydrous tetrahydro furan (THF) in the presence of DCC and N-hydroxy benzo triazole (HOBt) 2(CH 2) 12CH 3
3) Boc-AA-OCH in hydrogenchloride-ethyl acetate solution 2(CH 2) 12CH 3Slough Boc and generate AA-O-CH 2(CH 2) 12CH 3
4) Boc-Pro-Ala in the presence of DCC and the HOBt in anhydrous THF with AA-OCH 2(CH 2) 12-CH 3Condensation is Boc-Pro-Ala-AA-OCH 2(CH 2) 12CH 3
5) Boc-Pro-Ala-AA-OCH in hydrogenchloride-ethyl acetate solution 2(CH 2) 12CH 3Remove Boc and generate Pro-Ala-AA-OCH 2(CH 2) 12CH 3
Three of the object of the invention is that above-mentioned compound is applied to prepare in the thrombolytic agent.
It is external that interior thrombus dissolving activity test shows that it is active that the compound 4a-r shown in the general formula I of the present invention has outstanding thrombus dissolving activity, can be applicable in the preparation of thrombolytic agent with body.
Description of drawings
Fig. 1 is the synthetic route chart of the compound 4a-r of I for general formula of the present invention;
I) DCC, HOBt, NMM; Ii) hydrogenchloride/ethyl acetate solution (4N); Iii) DCC, HOSu, NaHCO 3And Ala; Iv) DCC, HOBt, NMM; V) hydrogenchloride/ethyl acetate solution (4N);
Fig. 2 is the transmission electron microscope photo of compound 4k.
The breviary term
The THF THF
HOBt N-hydroxy benzo triazole
DCC dicyclohexyl carbonyl diimine
The DCU NSC 30023
The TLC thin-layer chromatography
The Boc tertbutyloxycarbonyl
Embodiment
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and are not to be understood that to be limitation of the present invention.
Embodiment 1 preparation Boc-Pro-Ala
2.00g (9.00mmol) Boc-Pro is dissolved with the anhydrous THF of 40ml.In the solution that obtains, add 1.16g (10.00mmol) N-hydroxy-succinamide (HOSu) and dissolving fully.After 10 minutes, under ice bath, add the solution of 2.29g (11.00mmol) DCC and the anhydrous THF of 20ml, obtain reaction solution.At room temperature stir 10h, thin-layer chromatography (Thin Layer Chromatography, TLC) (methylene chloride, 30: 1)) shows that Boc-Pro disappears.Filtering NSC 30023 (DCU), THF is removed in decompression.Residue is used the 150ml acetic acid ethyl dissolution, and the solution that obtains is used 5%NaHCO successively 3It is inferior that the aqueous solution is washed, the saturated NaCl aqueous solution is respectively given a baby a bath on the third day after its birth, and organic phase is evaporated to dried.Residuum obtains reaction solution (I) with 40ml THF dissolving.827mg (9mmol) Ala is used the 30ml water dissolution, obtain reaction solution (II).Under stirring reaction solution (II) is added in the reaction solution (I), use saturated NaHCO 3Transfer pH of mixed between the 8-9, stir 16h under the room temperature, take out CO in the reaction process constantly 2Gas, and keep pH value of solution between the 8-9, TLC (methylene chloride, 30: 1) shows that reaction solution (I) disappears.Reaction back solution decompression is removed THF, and residual solution is used saturated KHSO 4Water is transferred solution PH to 2, and the solution that obtains is collected organic phase with ETHYLE ACETATE repeatedly extraction on a small quantity, and organic layer is given a baby a bath on the third day after its birth inferior with the saturated NaCl aqueous solution, tell organic phase and use anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, get the target compound of 1.99g (66%) present embodiment, are the colorless solid powder.ESI-MS(m/e)：309[M+Na] +
Embodiment 2 preparation Boc-Gly-OCH 2(CH 2) 12CH 3(1a)
828mg (4.60mmol) Boc-Gly is dissolved with the anhydrous THF of 20ml.In the solution that obtains, add 0.68g (4.80mmol) HOBt.After 10 minutes, under ice bath, add the solution of 1.13g (5.50mmo1) NSC 57182 (DCC) and the anhydrous THF of 25ml, obtain reaction solution (I).1.00g (4.60mmo1) CH 3(CH 2) 12CH 2OH is dissolved in the anhydrous THF of 10ml and stirred 30 minutes, obtains reaction solution (II).Under the ice bath reaction solution (II) is added in the reaction solution (I), stirring at room 6h then, TLC (sherwood oil: acetone, 4: 1) shows that Boc-Gly disappears.Reaction mixture filters, filtering DCU.Filtrate decompression concentrates, and removes THF.Residue is used the 100ml acetic acid ethyl dissolution.The solution that obtains is used saturated NaHCO successively 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed, 5%KHSO 4The aqueous solution is washed with the saturated NaCl aqueous solution and is washed.Ethyl acetate solution is used anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, and residue obtains the target compound of 582mg (46%) present embodiment through column chromatography purification, is colourless powder.ESI-MS(m/e)：372[M+H] +.
Embodiment 3 preparation Boc-Ala-OCH 2(CH 2) 12CH 3(1b)
According to the method for embodiment 2 by 895mg (4.70mmol) Boc-Ala and 1.00g (4.60mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 899mg (50%) present embodiment, is the colorless solid powder.ESI-MS(m/e)：408[M+Na] +
Embodiment 4 preparation Boc-Phe-OCH 2(CH 2) 12CH 3(1c)
According to the method for embodiment 2 by 1.35g (4.70mmol) Boc-Phe and 1.00g (4.60mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 1.12g (52%) present embodiment, is the colorless solid powder.ESI-MS(m/e)：462[M+H] +
Embodiment 5 preparation Boc-Val-OCH 2(CH 2) 12CH 3(1d)
According to the method for embodiment 2 by 1.02g (4.70mmol) Boc-Val and 1.00g (4.60mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 1.12mg (58%) present embodiment, is colorless oil.ESI-MS(m/e)：413[M+H] +
Embodiment 6 preparation Boc-Leu-OCH 2(CH 2) 12CH 3(1e)
According to the method for embodiment 2 by 1.34g (4.70mmol) Boc-Leu and 1.00g (4.60mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 957mg (48%) present embodiment, is colorless oil.ESI-MS(m/e)：450[M+Na] +
Embodiment 7 preparation Boc-Ile-OCH 2(CH 2) 12CH 3(1f)
According to the method for embodiment 2 by 1.33g (4.70mmol) Boc-Ile and 1.00g (4.60mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 997mg (50%) present embodiment, is colorless oil.ESI-MS(m/e)：450[M+Na] +
Embodiment 8 preparation Boc-Pro-OCH 2(CH 2) 12CH 3(1g)
According to the method for embodiment 2 by 1.10g (4.70mmol) Boc-Pro and 1.00g (4.60mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 998mg (52%) present embodiment, is yellow oil.ESI-MS(m/e)：434[M+Na] +
Embodiment 9 preparation Boc-Tyr-OCH 2(CH 2) 12CH 3(1h)
According to the method for embodiment 2 by 2.42g (4.70mmol) Boc-Tyr and 1.00g (4.60mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 1.00g (45%) present embodiment, is colorless oil.ESI-MS(m/e)：500[M+Na] +
Embodiment 10 preparation Boc-Trp-OCH 2(CH 2) 12CH 3(1i)
According to the method for embodiment 2 by 2.57g (4.70mmol) Boc-Trp and 1.00g (4.60mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 1.05g (45%) present embodiment, is colorless oil.ESI-MS(m/e)：501[M+H] +
Embodiment 11 preparation Boc-Asp (OBzl)-OCH 2(CH 2) 12CH 3(1j)
According to the method for embodiment 2 by 2.56g (7.90mmol) Boc-Asp (OBzl) and 1.50g (7.00mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 1.38g (38%) present embodiment, is the colorless solid powder.ESI-MS(m/e)：520[M+H] +
Embodiment 12 preparation Boc-Glu (OBzl)-OCH 2(CH 2) 12CH 3(1k)
According to the method for embodiment 2 by 3.14g (9.30mmol) Boc-Glu (OBzl) and 2.00g (9.30mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 1.69g (34%) present embodiment, is the colorless solid powder.ESI-MS(m/e)：556[M+Na] +
Embodiment 13 preparation Boc-Arg (NO 2)-OCH 2(CH 2) 12CH 3(1l)
According to the method for embodiment 2 by 3.02g (9.50mmol) Boc-Arg (NO 2) and 1.97g (9.30mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 822mg (17%) present embodiment, is the colorless solid powder.ESI-MS(m/e)：538[M+Na] +
Embodiment 14 preparation Boc-Ser (Bzl)-OCH 2(CH 2) 12CH 3(1m)
According to the method for embodiment 2 by 2.84g (9.60mmol) Boc-Ser (Bzl) and 1.80g (8.40mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 1.46g (51%) present embodiment, is colorless oil.ESI-MS(m/e)：514[M+Na] +
Embodiment 15 preparation Boc-Thr (Bzl)-OCH 2(CH 2) 12CH 3(1n)
According to the method for embodiment 2 by 2.40g (7.70mmol) Boc-Thr (Bzl) and 1.50g (7.00mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 1.32g (36%) present embodiment, is colorless oil.ESI-MS(m/e)：528[M+Na] +
Embodiment 16 preparation Boc-Lys (Z)-OCH 2(CH 2) 12CH 3(1o)
According to the method for embodiment 2 by 3.55g (9.30mmol) Boc-Lys (Z) and 2.00g (9.30mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 2.11g (39%) present embodiment, is colorless oil.ESI-MS(m/e)：599[M+Na] +
Embodiment 17 preparation Boc-Met-OCH 2(CH 2) 12CH 3(1p)
According to the method for embodiment 2 by 2.22g (8.90mmol) Boc-Met and 1.90g (9.30mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 2.41g (61%) present embodiment, is colorless oil.ESI-MS(m/e)：468[M+Na] +
Embodiment 18 preparation Boc-Asn-OCH 2(CH 2) 12CH 3(1q)
According to the method for embodiment 2 by 2.16g (9.30mmol) Boc-Asn and 2.00g (9.30mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 1.60g (40%) present embodiment, is colorless oil.ESI-MS(m/e)：451[M+Na] +
Embodiment 19 preparation Boc-Gln-OCH 2(CH 2) 12CH 3(1r)
According to the method for embodiment 2 by 2.29g (9.30mmol) Boc-Gln and 2.00g (9.30mmol) CH 3(CH 2) 12CH 2OH, the process column chromatography makes the target compound of 1.58g (38%) present embodiment, is colorless oil.ESI-MS(m/e)：443[M+H] +
Embodiment 20 preparation Gly-OCH 2(CH 2) 12CH 3(2a)
1.28g (3.45mmol) is dissolved in 15ml 4N hydrogenchloride-ethyl acetate solution, stirring at room 3 hours, TLC (sherwood oil/acetone, 4: 1) shows Boc-Gly-OCH 2(CH 2) 12CH 3Disappear, concentrating under reduced pressure is removed ETHYLE ACETATE, and residue adds a small amount of ether repeatedly and carries out concentrating under reduced pressure to remove de-chlorine hydride.Add a small amount of ether at last residue is ground to form the target compound of 900mg (84%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：272[M+H] +.
Embodiment 21 preparation Ala-OCH 2(CH 2) 12CH 3(2b)
Method 328mg (0.85mmol) Boc-Ala-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 270mg (99%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：286[M+H] +..
Embodiment 22 preparation Phe-OCH 2(CH 2) 12CH 3(2c)
Method 1.09g (2.36mmol) Boc-Phe-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 935mg (99%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：362[M+H] +.
Embodiment 23 preparation Val-OCH 2(CH 2) 12CH 3(2d)
Method 927mg (2.24mmol) Boc-Val-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 770mg (98%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：314[M+H] +.
Embodiment 24 preparation Leu-OCH 2(CH 2) 12CH 3(2e)
Method 360mg (0.84mmol) Boc-Leu-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 280mg (92%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：328[M+H] +.
Embodiment 25 preparation Ile-OCH 2(CH 2) 12CH 3(2f)
Method 1.60g (3.74mmol) Boc-Ile-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 1.30g (99%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：328[M+H] +.
Embodiment 26 preparation Pro-OCH 2(CH 2) 12CH 3(2g)
Method 2.00g (4.86mmol) Boc-Pro-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 1.48g (88%) present embodiment, be the yellow solid powder.ESI-MS(m/e)：312[M+H] +.
Embodiment 27 preparation Tyr-OCH 2(CH 2) 12CH 3(2h)
Method 1.06g (2.22mmol) Boc-Tyr-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 910mg (98%) present embodiment, be pale brown look pressed powder.ESI-MS(m/e)：378[M+H] +.
Embodiment 28 preparation Trp-OCH 2(CH 2) 12CH 3(2i)
Method 1.32g (2.64mmol) Boc-Trp-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 1.12g (99%) present embodiment, be the gray solid powder.ESI-MS(m/e)：401[M+H] +.
Embodiment 29 preparation Asp (OBzD-OCH 2(CH 2) 12CH 3(2j)
Method 1.40g (2.69mmol) Boc-Asp (OBzl)-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 1.17g (98%) present embodiment, be colorless oil.ESI-MS(m/e)：420[M+H] +.
Embodiment 30 preparation Glu (OBzD-OCH 2(CH 2) 12CH 3(2k)
Method 1.70g (3.18mmol) Boc-Glu (OBzl)-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 1.40g (93%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：434[M+H] +.
Embodiment 31 preparation Arg (N0 2)-OCH 2(CH 2) 12CH 3(2l)
Method 800mg (1.55mmol) Boc-Arg (NO according to embodiment 20 2)-OCH 2(CH 2) 12CH 3Make the target compound of 650mg (91%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：416[M+H] +.
Embodiment 32 preparation Ser (Bzl)-OCH 2(CH 2) 12CH 3(2m)
Method 2.20g (4.48mmol) Boc-Ser (Bzl)-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 1.80g (95%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：392[M+H] +.
Embodiment 33 preparation Thr (Bzl)-OCH 2(CH 2) 12CH 3(2n)
Method 1.30g (2.57mmol) Boc-Thr (Bzl)-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 1.09g (96%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：406[M+H] +.
Embodiment 34 preparation Lys (Z)-OCH 2(CH 2) 12CH 3(2o)
Method 1.10g (1.90mmol) Boc-Lys (Z)-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 960mg (98%) present embodiment, be colorless oil.ESI-MS(m/e)：477[M+H] +.
Embodiment 35 preparation Met-OCH 2(CH 2) 12CH 3(2p)
Method 3.19g (7.16mmol) Boc-Met-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 2.50g (94%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：346[M+H] +.
Embodiment 36 preparation Asn-OCH 2(CH 2) 12CH 3(2q)
Method 1.10g (2.57mmol) Boc-Asn-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 928mg (99%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：329[M+H] +.
Embodiment 37 preparation Gln-OCH 2(CH 2) 12CH 3(2r)
Method 1.57g (3.55mmol) Boc-Gln-OCH according to embodiment 20 2(CH 2) 12CH 3Make the target compound of 1.20g (90%) present embodiment, be the colorless solid powder.ESI-MS(m/e)：343[M+H] +.
Embodiment 38 preparation Boc-Pro-Ala-Gly-OCH 2(CH 2) 12CH 3(3a)
According to the method for embodiment 2 by 228mg (0.80mmol) Boc-Pro-Ala and 255mg (0.80mmol) Gly-OCH 2(CH 2) 12CH 3, make 94mg (22%) target compound through column chromatography, be colorless oil.[α] D 25＝-163.1(c＝0.50，CH 3OH)；ESI-MS(m/e)：563[M+Na+H] +；IR(KBr)：3859，3606，3059，2865，2743，2522，2227，2044，1744，1673，1549，1204，1085，968，848，475cm -1； 1H-NMR(300MHz，CDCl 3)：δ/ppm＝4.58-4.48(m，1H)，4.27(s，1H)，4.14-4.10(d，J＝6.6Hz，2H)，4.02-3.89(m，2H)，3.46(s，2H)，2.18(s，2H)，1.90(s，1H)，1.65-1.60(m，2H)，1.46(s，9H)，1，41-1.39(d，J＝7.2Hz，3H)，1.26(s，22H)，0.90-0.86(t，J＝4.8Hz，3H).
Embodiment 39 preparation Boc-Pro-Ala-Ala-OCH 2(CH 2) 12CH 3(3b)
According to the method for embodiment 2 by 918mg (3.20mmol) Boc-Pro-Ala and 1.00g (3.20mmol) Ala-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 656mg (37%) present embodiment, is colorless solid.Mp：65-66℃；[α] D 25＝-81.4(c＝0.50，CH 3OH)；ESI-MS(m/e)：577[M+Na+H] +；IR(KBr)：3840，3744，3292，3077，2921，2852，2360，1742，1702，1646，1547，1457，1398，1209，1164，964，918，877，768，723，671cm -1； 1H-NMR(300MHz，CDCl 3)：δ/ppm＝4.58-4.44(m，2H)，4.30(s，1H)，4.20-4.07(m，2H)，3.47-3.39(m，2H)，2.27-2.14(m，1H)，1.90(s，2H)，1.79(s，1H)，1.66-1.62(m，2H)，1.47(s，9H)，1.43-1.38(m，6H)，1.27-1.16(m，22H)，0.91-0.87(m，3H).
Embodiment 40 preparation Boc-Pro-Ala-Phe-OCH 2(CH 2) 12CH 3(3c)
According to the method for embodiment 2 by 675mg (2.30mmol) Boc-Pro-Ala and 940mg (2.30mmol) Phe-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 742mg (50%) present embodiment, is colorless solid.Mp：74-75℃；[α] D 25＝-57.2(c＝0.50，CH 3OH)；ESI-MS(m/e)：652[M+Na] +；IR(KBr)：3306，2923，2854，2361，1741，1648，1540，1457，1395，1201cm -1； 1H-NMR(300MHz，CDCl 3)：δ/ppm＝7.37-7.23(s，3H)，7.14-7.11(d，J＝7.2Hz，2H)，4.85-4.79(m，1H)，4.42-4.38(m，1H)，4.23(s，1H)，4.11-4.07(t，J＝6.6Hz，2H)，3.45(s，2H)，3.14-3.10(m，2H)，2.19-2.12(m，1H)，1.90(s，2H)，1.71(s，1H)，1.61-1.59(m，2H)，1.48(s，9H)，1.34-1.32(m，3H)，1.25(s，22H)，0.92-0.87(t，J＝6.0Hz，3H).
Embodiment 41 preparation Boc-Pro-Ala-Val-OCH 2(CH 2) 12CH 3(3d)
According to the method for embodiment 2 by 623mg (2.10mmol) Boc-Pro-Ala and 780mg (2.10mmol) Val-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 772mg (61%) present embodiment, is colorless oil.[α] D 25＝-90.5(c＝0.50，CH 3OH)；ESI-MS(m/e)：582[M+H] +；IR(KBr)：3816，3338，2945，2833，2659，2309，2145，1948，1740，1676，1556，1455，1258，1165，1114，1031，673cm -1； 1H-NMR(300MHz，CDCl 3)：δ/ppm＝4.52-4.48(m，2H)，4.29(s，1H)，4.23-4.06(m，2H)，3.46(s，2H)，2.22-2.15(m，2H)，1.89(s，2H)，1.81(s，1H)，1.66-1.62(m，2H)，1.46(s，9H)，1.40-1.37(d，J＝6.6Hz，3H)，1.27(s，22H)，0.95-0.87(m，9H).
Embodiment 42 preparation Boc-Pro-Ala-Leu-OCH 2(CH 2) 12CH 3(3e)
According to the method for embodiment 2 by 243mg (0.80mmol) Boc-Pro-Ala and 306mg (0.80mmol); Leu-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 278mg (55%) present embodiment, is colorless oil.[α] D 25＝-123.3(c＝0.50，CH 3OH)；ESI-MS(m/e)：619[M+Na+H] +；IR(KBr)：3852，3591，3060，2865，2753，2522，2228，2045，1666，1525，1335，1147，1086，977，830，540cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝4.57-4.50(m，2H)，4.27(s，1H)，4.15-4.03(m，2H)，3.46-3.37(m，2H)，2.19(s，1H)，1.88-1.87(m，2H)，1.66-1.51(m，5H)，1.44(s，9H)，1.37(s，2H)，1.35(s，2H)，1.25(s，22H)，0.92-0.89(m，6H)，0.87-0.84(m，3H).
Embodiment 43 preparation Boc-Pro-Ala-Ile-OCH 2(CH 2) 12CH 3(3f)
According to the method for embodiment 2 by 1.07g (3.70mmol) Boc-Pro-Ala and 1.36g (3.70mmol) Ile-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 1.06g (48%) present embodiment, is colorless solid.Mp：57-58℃；[α] D 25＝-55.4(c＝0.50，CH 3OH)；ESI-MS(m/e)：618[M+Na] +；IR(KBr)：3743，3307，2962，2923，2853，2360，1750，1700，1652，1542，1460，1398，1254，1196，1163，1124，976，645cm -1； 1H-NMR(300MHz，CDCl 3)：δ/ppm＝4.55-4.50(m，2H)，4.28(s，1H)，4.18-4.03(m，3H)，3.47-3.36(m，2H)，2.17(s，2H)，1.89-1.87(m，3H)，1.65-1.58(m，2H)，1.45(s，9H)，1.37-1.35(d，J＝6.9Hz，4H)，1.20(s，22H)，0.92-0.85(m，9H).
Embodiment 44 preparation Boc-Pro-Ala-Pro-OCH 2(CH 2) 12CH 3(3g)
According to the method for embodiment 2 by 501mg (1.70mmol) Boc-Pro-Ala and 800mg (1.70mmol) Pro-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 535mg (45%) present embodiment, is yellow oil.[α] D 25＝-69.9(c＝0.50，CH 3OH)；ESI-MS(m/e)：602[M+Na] +；IR(KBr)：3395，3301，2943，2831，2326，2134，1679，1451，1417，1171，1114，1030，655cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝4.72(s，1H)，4.53-4.50(m，1H)，4.26(s，1H)，4.13-4.03(m，1H)，3.69-3.62(m，2H)，3.48-3.37(m，2H)，3.17(s，4H)，2.06-2.02(m，4H)，1.90-1.86(m，2H)，1.62-1.55(m，2H)，1.45(s，9H)，1.38-1.36(d，J＝6.6Hz，3H)，1.24(s，20H)，0.90-0.86(t，J＝6.0Hz，3H).
Embodiment 45 preparation Boc-Pro-Ala-Tyr-OCH 2(CH 2) 12CH 3(3h)
According to the method for embodiment 2 by 637mg (2.20mmol) Boc-Pro-Ala and 920mg (2.20mmol) Tyr-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 890mg (62%) present embodiment, is colorless solid.Mp：98-99℃；[α] D 25＝-93.8(c＝0.50，CH 3OH)；ESI-MS(m/e)：647[M+2H] +；IR(KBr)：3745，3295，3095，2925，2855，2361，1705，1653，1542，1455，1403，1367，1293，1254，1229，1168，1123，827，714cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝7.31-7.28(m，1H)，6.95-6.92(d，J＝8.4Hz，2H)，6.74-6.71(d，J＝8.4Hz，2H)，4.81-4.75(m，1H)，4.41(s，1H)，4.25(s，1H)，4.13-4.08(t，J＝6.6Hz，2H)，3.46-3.37(m，2H)，3.10-2.96(m，2H)，2.19(s，2H)，1.87(s，2H)，1.63-1.55(m，3H)，1.46(s，9H)，1.26(s，24H)，0.91-0.86(t，J＝6.2Hz，3H).
Embodiment 46 preparation Boc-Pro-Ala-Trp-OCH 2(CH 2) 12CH 3(3i)
According to the method for embodiment 2 by 747mg (2.60mmol) Boc-Pro-Ala and 1.15g (2.60mmol) Trp-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 994mg (57%) present embodiment, is yellow oil.[α] D 25＝-116.8(c＝0.50，CH 3OH)；ESI-MS(m/e)：669[M+H] +；IR(KBr)：3456，2940，2831，2523，1673，1450，1113，1031，660cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝8.37(s，1H)，7.54-7.52(d，J＝7.8Hz，1H)，7.36-7.33(d，J＝7.8Hz，1H)，7.28(s，1H)，7.20-7.08(m，2H)，7.03(s，1H)，4.89-4.87(m，1H)，4.44-4.40(m，1H)，4.17(s，1H)，4.17-3.98(m，2H)，3.45(s，2H)，3.31-3.29(d，J＝5.4Hz，2H)，2.19-2.11(m，1H)，1.86(s，1H)，1.77(s，1H)，1.62-1.56(m，3H)，1.48(s，9H)，1.17(s，24H)，0.92-0.87(d，J＝6.6Hz，3H).
Embodiment 47 preparation Boc-Pro-Ala-Asp (OBzl)-OCH 2(CH 2) 12CH 3(3j)
According to the method for embodiment 2 by 627mg (2.20mmol) Boc-Pro-Ala and 1.00g (2.20mmol) Asp (OBzl)-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 602mg (40%) present embodiment, is colorless solid.Mp：51-52℃；[α] D 25＝-78.3(c＝0.50，CH 3OH)；ESI-MS(m/e)：711[M+Na+H] +；IR(KBr)：3360，2924，2854，2360，1737，1699，1651，1516，1457，1401，1364，1297，1168，1121，980，747，592cm -1； 1H-NMR(300MHz，CDCl 3)：δ/ppm＝7.34-7.28(d，J＝18.6Hz，5H)，5.11(s，2H)，4.83-4.82(d，J＝3.6Hz，1H)，4.44(s，1H)，4.26(s，1H)，4.10-4.05(t，J＝6.6Hz，2H)，3.45(s，2H)，3.01-2.84(m，2H)，2.25-2.10(m，2H)，1.88-1.86(m，2H)，1.56-1.54(m，2H)，1.45(s，9H)，1.38-1.36(d，J＝6.6Hz，3H)，1.25(s，22H)，0.89-0.85(t，J＝6.0Hz，3H).
Embodiment 48 preparation Boc-Pro-Ala-Glu (OBzl)-OCH 2(CH 2) 12CH 3(3k)
According to the method for embodiment 2 by 851mg (2.90mmol) Boc-Pro-Ala and 1.40g (2.90mmol) Glu (OBzl)-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 938mg (45%) present embodiment, is colorless oil.[α] D25＝-117.4(c＝0.50，CH 3OH)；ESI-MS(m/e)：724[M+Na]；IR(KBr)：3564，3061，2862，2781，2598，2520，2227，2045，1739，1675，1548，1374，1168，993，748，583cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝7.35-7.28(m，5H)，5.11(s，2H)，4.60-4.53(m，1H)，4.46(s，1H)，4.28(s，1H)，4.13-4.09(t，J＝6.6Hz，2H)，3.44-3.35(m，2H)，2.61-2.35(m，2H)，2.28-2.17(m，2H)，2.12-1.96(m，2H)，1.87-1.85(m，2H)，1.64-1.54(m，2H)，1.44(s，9H)，1.38-1，36(d，J＝7.2Hz，3H)，1.26(s，22H)，0.90-0.86(t，J＝6.3Hz，3H).
Embodiment 49 preparation Boc-Pro-Ala-Arg (NO 2)-OCH 2(CH 2) 12CH 3(3l)
According to the method for embodiment 2 by 443mg (1.50mmol) Boc-Pro-Ala and 700mg (1.50mmol) Arg (NO 2)-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 556mg (50%) present embodiment, is colorless solid.Mp：59-60℃；[α] D 25＝-101.8(c＝0.50，CH 3OH)；ESI-MS(m/e)：684[M+H] +；IR(KBr)：3739，3306，2925，2855，2360，1742，1654，1540，1455，1399，1258，1164，1119，976，774，724，662cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝8.71(s，1H)，7.88(s，1H)，4.56-4.48(m，2H)，4.28(s，1H)，4.14-4.10(t，J＝6.6Hz，2H)，3.94(s，1H)，3.44-3.30(m，4H)，2.18-2.08(m，2H)，1.90-1.88(m，4H)，1.65-1.63(m，6H)，1.46(s，9H)，1.40-1.38(d，J＝6.3Hz，3H)，1.25(s，22H)，0.90-0.85(t，J＝6.3Hz，3H).
Embodiment 50 preparation Boc-Pro-Ala-Ser (Bzl)-OCH 2(CH 2) 12CH 3(3m)
According to the method for embodiment 2 by 1.28g (4.40mmo1) Boc-Pro-Ala and 1.90g (4.40mmol) Ser (Bzl)-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 1.33g (48%) present embodiment, is yellow oil.[α] D 25＝-67.3(c＝0.50，CH 3OH)；ESI-MS(m/e)：660[M+H] +IR(KBr)：3504，3191，2831，2602，2522，2227，2044，1744，1675，1451，1114，1033，649cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝7.37-7.26(m，5H)，4.72-4.68(m，1H)，4.57-4.45(m，3H)，4.27(s，1H)，4.19-4.07(m，2H)，3.92-3.86(m，1H)，3.71-3.65(m，1H)，3.45(s，2H)，2.21-2.17(m，1H)，2.00(s，1H)，1.89-1.84(m，2H)，1.62-1.56(m，2H)，1.46(s，9H)，1.41-1.36(m，3H)，1.24(s，22H)，0.91-0.86(t，J＝6.6Hz，3H).
Embodiment 51 preparation Boc-Pro-Ala-Thr (Bzl)-OCH 2(CH 2) 12CH 3(3n)
According to the method for embodiment 2 by 712mg (2.50mmol) Boc-Pro-Ala and 1.10g (2.50mmol) Thr (Bzl)-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 759mg (46%) present embodiment, is colorless solid.Mp：49-50℃；[α] D 25＝-95.4(c＝0.50，CH 3OH)；ESI-MS(m/e)：674[M+H] +；IR(KBr)：3745，3304，2923，2854，2360，1739，1702，1652，1540，1456，1399，1212，1166，1121，1074，469cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝7.36-7.20(m，5H)，4.68-4.59(m，2H)，4.55-4.51(m，1H)，4.40-4.36(d，J＝11.7Hz，1H)，4.27-3.98(m，4H)，3.46(s，2H)，2.17(s，1H)，2.01(s，1H)，1.87-1，86(m，2H)，1.58-1.51(m，2H)，1.45(s，9H)，1.43-1.40(m，3H)，1.26(s，22H)，1.22-1.20(m，3H)，0.91-0.86(t，J＝6.6Hz，3H).
Embodiment 52 preparation Boc-Pro-Ala-Iys (Z)-OCH 2(CH 2) 12CH 3(3o)
According to the method for embodiment 2 by 529mg (1.80mmol) Boc-Pro-Ala and 950mg (1.80mmol) Lys (Z)-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 400mg (29%) present embodiment, is yellow oil.[α] D 25＝-28.2(c＝0.50，CH 3OH)；ESI-MS(m/e)：767[M+Na] +；IR(KBr)：3859，3525，2590，2521，2225，2044，1677，1420，1262，996，597cm -1 1H-NMR(300MHz，CDCl 3)：δ/ppm＝7.36-7.30(m，5H)，5.14-5.04(m，2H)，4.55-4.48(m，2H)，4.31-4.27(m，1H)，4.14-4.09(t，J＝6.6Hz，2H)，3.91-3.88(m，1H)，3.42(s，1H)，3.27-3.11(m，2H)，2.18(s，1H)，1.85-1.80(m，4H)，1.72-1.61(m，4H)，1.59-1.49(m，2H)，1.46(s，9H)，1.37-1.35(d，J＝6.9HZ，5H)，1.26(s，22H)，0.91-0.86(t，J＝6.6Hz，3H).
Embodiment 53 preparation Boc-Pro-Ala-Met-OCH 2(CH 2) 12CH 3(3p)
According to the method for embodiment 2 by 1.87g (6.50mmol) Boc-Pro-Ala and 2.50g (6.50mmol) Met-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 2.60g (65%) present embodiment, is yellow oil.[α] D 25＝-22.6(c＝0.50，CH 3OH)；ESI-MS(m/e)：652[M+K] +；IR(KBr)：3859，3563，3O94，2884，2783，2594，2522，2227，2044，1732，1671，1483，1120，987，740cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝4.64(s，1H)，4.44-4.39(t，J＝6.9Hz，1H)，4.31-4.28(m，1H)，4.16-4.11(t，J＝6.6Hz，2H)，3.45(s，2H)，2.89-2.68(m，2H)，2.59(s，3H)，2.42-2.36(m，1H)，2.23-2.11(m，2H)，2.04-2.04(m，1H)，1.91-1.87(m，2H)，1.66-1.62(t，J＝6.6Hz，2H)，1.47(s，9H)，1.39-1.37(d，J＝7.2Hz，3H)，1.26(s，22H)，0.90-0.86(t，J＝6.6Hz，3H).
Embodiment 54 preparation Boc-Pro-Ala-Asn-OCH 2(CH 2) 12CH 3(3q)
According to the method for embodiment 2 by 780mg (2.70mmol) Boc-Pro-Ala and 981mg (2.70mmol) Asn-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 345mg (21%) present embodiment, is colorless solid.Mp：80-81℃；[α] D 25＝-27.8(c＝0.50，CH 3OH)；ESI-MS(m/e)：619[M+Na] +；IR(KBr)：3420，3298，3219，2921，2852，2361，1742，1667，1513，1404，1211，1164，1119，1053，604cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝7.57-7.54(d，J＝8.1Hz，1H)，6.57(s，1H)，6.35(s，1H)，4.91(s，1H)，4.62(s，1H)，4.23(s，1H)，4.14-4.09(d，J＝6.6Hz，2H)，3.47-3.40(m，2H)，2.75(s，2H)，2.12-2.10(m，2H)，1.91-1.86(m，2H)，1.64-1.60(m，2H)，1.46(s，9H)，1.41-1.38(d，J＝6.9Hz，3H)，1.25(s，22H)，0.90-0.86(t，J＝6.6Hz，3H).
Embodiment 55 preparation Boc-Pro-Ala-Gln-OCH 2(CH 2) 12CH 3(3r)
According to the method for embodiment 2 by 981mg (3.40mmol) Boc-Pro-Ala and 1.30g (3.40mmol) Gln-OCH 2(CH 2) 12CH 3, the process column chromatography makes the target compound of 600mg (28%) present embodiment, is colorless solid.Mp：90-91℃；[α] D 25＝-40.1(c＝0.50，CH 3OH)；ESI-MS(m/e)：633[M+Na] +；IR(KBr)：3424，3289，3203，3090，2924，2853，2360，1728，1660，1550，1404，1314，1252，1213，1168，1124，989，662cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝7.11(s，1H)，6.68(s，1H)，5.78(s，1H)，4.60-4.47(m，2H)，4.31(s，1H)，4.13-4.09(t，J＝6.9Hz，2H)，3.47-3.39(m，2H)，2.27-2.25(m，4H)，2.10-2.00(m，2H)，1.94-1.86(m，2H)，1.65-1.60(m，2H)，1.46(s，9H)，1.38-1.36(d，J＝6.9Hz，3H)，1.26(s，22H)，0.90-0.86(t，J＝6.6Hz，3H).
Embodiment 56 preparation Boc-Pro-Ala-Asp-OCH 2(CH 2) 12CH 3
With 320mg (0.46mmol) Boc-Pro-Ala-Asp (OBzl)-OCH 2(CH 2) 12CH 3Place the 50ml eggplant-shape bottle, use dissolve with methanol, add 50mg Pd/C (15%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried, get 270mg (97%) title compound, are colorless oil.ESI-MS(m/e)：597[M-H] -.
Embodiment 57 preparation Boc-Pro-Ala-Glu-OCH 2(CH 2) 12CH 3
With 420mg (0.59mmol) Boc-Pro-Ala-Glu (OBzl)-OCH 2(CH 2) 12CH 3Place the 50ml eggplant-shape bottle, use dissolve with methanol, add 65mg Pd/C (15%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried, get 350mg (95%) title compound, are colorless oil.ESI-MS(m/e)：610[M-H] -.
Embodiment 58 preparation Boc-Pro-Ala-Arg-OCH 2(CH 2) 12CH 3
With 560mg (0.82mmol) Boc-Pro-Ala-Arg (NO 2)-OCH 2(CH 2) 12CH 3Place the 50ml eggplant-shape bottle, use dissolve with methanol, add 90mg Pd/C (15%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried, get 510mg (97%) title compound, are colorless oil.ESI-MS(m/e)：639[M+H] +
Embodiment 59 preparation Boc-Pro-Ala-Ser-OCH 2(CH 2) 12CH 3
With 390mg (0.59mmol) Boc-Pro-Ala-Ser (Bzl)-OCH 2(CH 2) 12CH 3Place the 50ml eggplant-shape bottle, use dissolve with methanol, add 60mg Pd/C (15%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried, get 330mg (98%) title compound, are colorless solid.ESI-MS(m/e)：592[M+Na] +
Embodiment 60 preparation Boc-Pro-Ala-Thr-OCH 2(CH 2) 12CH 3
With 380mg (0.56mmol) Boc-Pro-Ala-Thr (Bzl)-OCH 2(CH 2) 12CH 3Place the 50ml eggplant-shape bottle, use dissolve with methanol, add 60mg Pd/C (15%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried, get 300mg (91%) title compound, are colorless oil.ESI-MS(m/e)：606[M+Na] +
Embodiment 61 preparation Boc-Pro-Ala-Iys-OCH 2(CH 2) 12CH 3
With 980mg (1.31mmol) Boc-Pro-Ala-Lys (Z)-OCH 2(CH 2) 12CH 3Place the 50ml eggplant-shape bottle, use dissolve with methanol, add 150mg Pd/C (15%), logical H 2(0.02Mba), stirring at room to raw material point disappears.Filtering Pd/C, filtrate decompression are concentrated into dried, get 750mg (93%) title compound, are colorless oil.ESI-MS(m/e)：611[M+H] +
Embodiment 62 preparation Pro-Ala-Gly-OCH 2(CH 2) 12CH 3(4a)
Method 200mg (0.37mmol) Boc-Pro-Ala-Gly-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 154mg (95%) present embodiment, be the yellow solid powder.Mp：119-120℃；[α] D 25＝-51.1(c＝0.50，CH 3OH)；ESI-MS(m/e)：440[M+H] +；IR(KBr)：3223，2924，2854，1749，1668，1551，1459，1394，1197cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝9.82(s，1H)，8.73(s，1H)，8.37(s，1H)，8.09(s，1H)，4.79(s，1H)，4.59(s，1H)，4.16-4.05(m，2H)，4.03-3.92(m，2H)，3.51(s，2H)，2.53(s，1H)，2.15(s，1H)，2.05-2.04(m，2H)，1.61-1.59(m，2H)，1.26(s，22H)，0.90-0.85(t，J＝6.6Hz，3H).
Embodiment 63 preparation Pro-Ala-Ala-OCH 2(CH 2) 12CH 3(4b)
Method 320mg (0.57mmol) Boc-Pro-Ala-Ala-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 264mg (94%) present embodiment, be the colorless solid powder.Mp：112-113℃；[α] D 25＝-103.0(c＝0.50，CH 3OH)；ESI-MS(m/e)：454[M+H] +；IR(KBr)：3286，3207，3053，2922，2853，1742，1656，1551，1458，1384，1340，1245，1209，1165，1049cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝10.66(s，1H)，8.63(s，1H)，8.30(s，1H)，4.82(s，2H)，4.45-4.40(m，1H)，4.21-4.13(m，1H)，4.07-3.99(m，1H)，3.48(s，2H)，2.54(s，1H)，2.18(s，1H)，2.06(s，3H)，1.64-1.61(m，2H)，1.50-1.48(m，6H)，1.27(s，22H)，0.91-0.87(t，J＝6.3Hz，3H).
Embodiment 64 preparation Pro-Ala-Phe-OCH 2(CH 2) 12CH 3(4c)
Method 300mg (0.47mmol) Boc-Pro-Ala-Phe-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 239mg (95%) present embodiment, be the colorless solid powder.Mp：97-99℃；[α] D 25＝-22.4(c＝0.50，CH 3OH)；ESI-MS(m/e)：530[M+H] +；IR(KBr)：3325，3206，3031，2919，2850，2365，1745，1687，1650，1537，1464，1347，1271，1179，1034，706，491cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝10.29(s，1H)，8.71(s，1H)，8.11(s，1H)，7.37-7.18(s，5H)，4.77-4.75(d，J＝6.0Hz，2H)，4.48(s，1H)，4.11-4.03(m，2H)，3.43(s，2H)，3.14(s，2H)，2.46(s，2H)，2.00(s，3H)，1.57(s，2H)，1.38(s，3H)，1.29(s，22H)，0.91-0.87(t，J＝6.0Hz，3H).
Embodiment 65 preparation Pro-Ala-Val-OCH 2(CH 2) 12CH 3(4d)
Method 320mg (0.55mmol) Boc-Pro-Ala-Val-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 254mg (96%) present embodiment, be the colorless solid powder.Mp：149-150℃；[α] D 25＝-105.7(c＝0.50，CH 3OH)；ESI-MS(m/e)：482[M+H] +；IR(KBr)：3297，3192，3058，2922，2852，2611，2484，2361，1735，1683，1640，1554，1467，1403，1269，1214，1160，661cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝10.86(s，1H)，8.67(s，1H)，8.28(s，2H)，5.03(s，1H)，4.87(s，1H)，4.33-4.29(m，1H)，4.21-4.13(m，1H)，4.05-3.98(m，1H)，3.47(s，2H)，2.54(s，1H)，2.31-2.25(m，1H)，2.05-2.02(m，3H)，1.63-1.61(s，2H)，1.46(s，3H)，1.26(s，22H)，1.06-1.02(t，J＝7.2Hz，6H)，0.90-0.86(t，J＝6.6Hz，3H).
Embodiment 66 preparation Pro-Ala-Leu-OCH 2(CH 2) 12CH 3(4e)
Method 330mg (0.55mmol) Boc-Pro-Ala-Leu-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 258mg (94%) present embodiment, be the colorless solid powder.Mp：102-103℃；[α] D 25＝-65.6(c＝0.50，CH 3OH)；ESI-MS(m/e)：496[M+H] +；IR(KBr)：3220，3051，2925，2856，2361，1744，1663，1550，1461，1385，1258，1195，1162，1043，970，668cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝10.88(s，1H)，8.92-8.17(m，2H)，5.12-4.83(m，1H)，4.30-4.14(m，2H)，3.93(s，1H)，3.49-3.44(m，1H)，2.64-2.50(m，1H)，2.04-1.90(m，4H)，1.62(s，3H)，1.51-1.44(m，2H)，1.27(s，24H)，0.99-0.82(m，9H).
Embodiment 67 preparation Pro-Ala-Ile-OCH 2(CH 2) 12CH 3(4f)
Method 350mg (0.58mmol) Boc-Pro-Ala-Ile-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 282mg (97%) present embodiment, be the colorless solid powder.Mp：143.3-144.0℃；[α] D 25＝-96.1(c＝0.50，CH 3OH)；ESI-MS(m/e)：496[M+H] +；IR(KBr)：3291，3189，3059，2953，2922，2853，2571，2481，2365，1735，1642，1556，1465，1412，1259，1210，1157，953，718，678cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝10.90(s，1H)，8.64-8.50(d，J＝40.5Hz，2H)，8.18(s，1H)，5.09(s，1H)，4.86(s，1H)，4.31(s，1H)，4.21-4.13(m，1H)，4.01-3.95(m，1H)，3.45(s，1H)，2.55(s，1H)，2.01(s，4H)，1.63-1.56(m，2H)，1.44(s，4H)，1.27(s，24H)，1.02-0.85(m，9H).
Embodiment 68 preparation Pro-Ala-Pro-OCH 2(CH 2) 12CH 3(4g)
Method 350mg (0.60mmol) Boc-Pro-Ala-Pro-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 272mg (94%) present embodiment, be the yellow solid powder.Mp：92-93℃；[α] D 25＝-126.0(c＝0.50，CH 3OH)；ESI-MS(m/e)：480[M+H] +；IR(KBr)：3204，3062，2924，2854，2646，2545，2434，2364，1742，1653，1558，1458，1352，1281，1179，1095，1047，927，721cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝10.68(s，1H)，8.23(m，1H)，8.18-8.16(m，1H)，4.67-4.65(m，2H)，4.52-4.48(m，1H)，4.15-4.00(m，2H)，3.73-3.68(m，1H)，3.62(s，1H)，3.50-3.46(m，2H)，2.47(s，1H)，2.23-2.16(m，1H)，2.04-1.93(m，6H)，1.61-1.57(m，2H)，1.43-1.41(d，J＝6.6Hz，3H)，1.24(s，22H)，0.89-0.84(t，J＝6.6Hz，3H).
Embodiment 69 preparation Pro-Ala-Tyr-OCH 2(CH 2) 12CH 3(4h)
Method 370mg (0.57mmol) Boc-Pro-Ala-Tyr-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 290mg (93%) present embodiment, be the colorless solid powder.Mp：109-110℃；[α] D 25＝-35.2(c＝0.50，CH 3OH)；ESI-MS(m/e)：546[M+H] +；IR(KBr)：3392，3327，3181，3038，2954，2917，2849，2756，2692，1739，1656，1558，1525，1445，1386，1350，1274，1213，1175，833，725，671，574，541，499cm -1. 1H-NMR(300MHz，DMSO-d6)：δ/ppm＝8.77-8.74(d，J＝7.5Hz，1H)，8.40-8.38(d，J＝7.5Hz，1H)，7.01-6.98(d，J＝8.1Hz，2H)，6.67-6.64(d，J＝8.4Hz，2H)，4.41-4.30(m，2H)，4.19-4.14(m，1H)，4.02-3.90(m，2H)，3.19-3.11(m，2H)，2.92-2.77(m，2H)，2.29-2.20(m，1H)，1.98-1.71(m，3H)，1.47(s，2H)，1.22(s，24H)，0.86-0.82(t，J＝6.6Hz，3H).
Embodiment 70 preparation Pro-Ala-Trp-OCH 2(CH 2) 12CH 3(4i)
Method 370mg (0.55mmol) Boc-Pro-Ala-Trp-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 298mg (95%) present embodiment, be the black solid powder.Mp：86-87℃；[α] D 25＝-31.2(c＝0.50，CH 3OH)；ESI-MS(m/e)：569[M+H] +；IR(KBr)：3226，3055，2924，2854，2361，1738，1665，1547，1458，1349，1208，741cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝9.73(s，1H)，8.57(s，1H)，8.20(s，1H)，7.61-7.45(m，1H)，7.35-7.33(m，1H)，7.28-7.25(m，1H)，7.09-7.01(m，2H)，4.76(s，1H)，4.53(s，1H)，4.03(s，2H)，3.28(s，3H)，2.19(s，1H)，2.09-2.05(m，1H)，1.67-1.61(m，3H)，1.27(s，24H)，0.91-0.87(t，J＝6.3Hz，3H).
Embodiment 71 preparation Pro-Ala-Asp-OCH 2(CH 2) 12CH 3(4j)
Method 250mg (0.36mmol) Boc-Pro-Ala-Asp-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 159mg (88%) present embodiment, be the colorless solid powder.Mp：107-108℃；[α] D 25＝-61.8(c＝0.50，CH 3OH)；ESI-MS(m/e)：498[M+H] +；IR(KBr)：3319，2921，2852，2365，1740，1653，1545，1461，1383，1270，1212，628cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝9.48(s，1H)，8.62(s，1H)，8.19-8.00(m，2H)，4.76-4.60(m，3H)，4.11(s，2H)，3.52(s，2H)，2.97-2.89(d，J＝24.3Hz，2H)，2.53(s，1H)，2.08-2.05(m，3H)，1.62(s，2H)，1.44(s，3H)，1.26(s，22H)，0.91-0.86(t，J＝6.6Hz，3H).
Embodiment 72 preparation Pro-Ala-Glu-OCH 2(CH 2) 12CH 3(4k)
Method 400mg (0.57mmol) Boc-Pro-Ala-Glu-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 245mg (85%) present embodiment, be the colorless solid powder.Mp：100-102℃；[α] D 25＝-122.0(c＝0.50，CH 3OH)；ESI-MS(m/e)：549[M+K] +；IR(KBr)：3291，3080，2921，2852，2744，2357，1741，1650，1554，1456，1387，1298，1252，1206cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝10.13-10.00(d，J＝41.1Hz，1H)，8.61(s，1H)，8.28-8.22(m，1H)，7.91(s，1H)，4.77-4.50(m，3H)，4.13-4.03(m，3H)，3.51(s，2H)，2.45(s，3H)，2.19(s，2H)，2.07-2.05(m，3H)，1.63(s，3H)，1.45(s，3H)，1.26(s，22H)，0.90-0.86(t，J＝6.6Hz，3H).
Embodiment 73 preparation Pro-Ala-Arg-OCH 2(CH 2) 12CH 3(4l)
Method 240mg (0.35mmol) Boc-Pro-Ala-Arg-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 147mg (80%) present embodiment, be the colorless solid powder.Mp：114-115℃；[α] D 25＝-61.2(c＝0.50，CH 3OH)；ESI-MS(m/e)：539[M+H] +；IR(KBr)：3163，3053，2925，2854，2357，1739，1661，1550，1457，1401，1252，1195，572cm -1. 1H-NMR(300MHz，DMSO-d6)：δ/ppm＝10.21-10.20(m，1H)，8.84-8.81(d，J＝6.9Hz，1H)，8.54-8.51(d，J＝7.2Hz，2H)，7.98(s，1H)，7.65-7.56(m，1H)，7.48-7.39(m，1H)，7.31-7.18(m，1H)，4.39-4.33(m，1H)，4.19-4.17(m，2H)，4.04-3.94(m，2H)，3.20-3.15(m，2H)，3.10(s，2H)，2.34-2.22(m，1H)，1.85(s，3H)，1.76-1.61(m，2H)，1.52(s，4H)，1.28-1.22(m，24H)，0.86-0.82(t，J＝6.6Hz，3H).
Embodiment 74 preparation Pro-Ala-Ser-OCH 2(CH 2) 12CH 3(4m)
Method 300mg (0.45mmol) Boc-Pro-Ala-Ser-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 178mg (%) present embodiment, be the colorless solid powder.Mp：128-129℃；[α] D 25＝-63.4(c＝0.50，CH 3OH)；ESI-MS(m/e)：470[M+H] +；IR(KBr)：3365，3334，2953，2921，2852，2736，1699，1740，1650，1551，1459，1395，1372，1276，1215，1154，1056，587cm -1. 1H-NMR(300MHz，DMSO-d6)：δ/ppm＝10.15(s，1H)，8.79-8.77(d，J＝7.2Hz，1H)，8.45-8.43(d，J＝7.5Hz，1H)，4.50-4.41(m，1H)，4.34-4.22(m，1H)，4.20-4.11(m，1H)，3.73-3.60(m，2H)，3.20-3.18(d，J＝6.0Hz，2H)，2.37-2.22(m，1H)，1.99-1.98(m，1H)，1.91-1.85(m，3H)，1.58-1.51(m，2H)，1.29-1.23(m，24H)，0.86-0.82(t，J＝6.6Hz，3H).
Embodiment 75 preparation Pro-Ala-Thr-OCH 2(CH 2) 12CH 3(4n)
Method 310mg (0.46mmol) Boc-Pro-Ala-Thr-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 190mg (83%) present embodiment, be the colorless solid powder.Mp：93-94℃；[α] D 25＝-63.4(c＝0.50，CH 3OH)；ESI-MS(m/e)：484[M+H] +；IR(KBr)：3221，3055，2925，2855，2361，1746，1671，1550，1459，1387，1308，1255，1205，1158，1102，1041cm -1. 1H-NMR(300MHz，DMSO-d6)：δ/ppm＝9.36-9.34(d，J＝7.5Hz，1H)，5.33-5.21(m，1H)，4.43-4.31(m，2H)，4.27-4.18(m，1H)，4.16-4.08(m，2H)，3.22-3.17(t，J＝6.6Hz，2H)，2.45-2.31(m，1H)，1.97-1.85(m，4H)，1.56-1.47(m，2H)，1.34-1.29(m，6H)，1.24(s，22H)，0.86-0.82(t，J＝6.6Hz，3H).
Embodiment 76 preparation Pro-Ala-Lys-OCH 2(CH 2) 12CH 3(4o)
Method 980mg (1.31mmol) Boc-Pro-Ala-Lys-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 500mg (68%) present embodiment, be the colorless solid powder.Mp：151-152℃；[α] D 25＝-32.3(c＝0.50，CH 3OH)；ESI-MS(m/e)：511[M+H] +；IR(KBr)：3438，2925，2279，1742，1666，1552，1460，1389，1254，1030，727cm -1. 1H-NMR(300MHz，CDCl 3)：δ/ppm＝9.77(s，1H)，9.08(s，1H)，8.61(s，1H)，8.10(s，1H)，4.67(s，1H)，4.47-4.41(m，1H)，4.07(s，2H)，3.59(s，1H)，3.43(s，1H)，3.08-2.97(m，1H)，2.56(s，1H)，2.07(s，4H)，1.85(s，4H)，1.62(s，4H)，1.45(s，5H)，1.26(s，24H)，0.90-0.86(t，J＝6.0Hz，3H).
Embodiment 77 preparation Pro-Ala-Met-OCH 2(CH 2) 12CH 3(4p)
Method 330mg (0.53mmo1) Boc-Pro-Ala-Met-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 280mg (95%) present embodiment, be the colorless solid powder.Mp：119-121℃；[α] D 25＝-47.8(c＝0.50，CH 3OH)；ESI-MS(m/e)：514[M+H] +；IR(KBr)：3294，3058，2925，2855，2271，1743，1651，1552，1457，1383，1214，1037，959，718cm -1. 1H-NMR?(300MHz，DMSO-d 6)：δ/ppm＝4.38-4.31(m，1H)，4.21-4.17(m，1H)，4.03-3.99(m，2H)，3.55(s，3H)，3.49-3.42(m，1H)，3.19-3.18(m，2H)，2.51-2.49(m，3H)，2.30-2.27(m，1H)，2.03(s，1H)，1.94-1.85(m，4H)，1.54-1.52(m，2H)，1.23(s，25H)，0.87-0.82(t，J＝6.6Hz，3H).
Embodiment 78 preparation Pro-Ala-Asn-OCH 2(CH 2) 12CH 3(4q)
Method 200mg (0.33mmol) Boc-Pro-Ala-Asn-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 150mg (84%) present embodiment, be the colorless solid powder.Mp：163-164℃；[α] D 25＝-57.7(c＝0.50，CH 3OH)；ESI-MS(m/e)：497[M+H] +；IR(KBr)：3305，2920，2850，2250，1742，1674，1555，1458，1398，1327，1270，1195，1026，995，724cm -1. 1H-NMR(300MHz，DMSO-d 6)：δ/ppm＝4.58-4.53(m，IH)，4.40-4.33(m，1H)，4.22-4.15(m，1H)，4.01-3.96(t，J＝6.3Hz，2H)，3.45(s，4H)，3.26-3.12(m，2H)，2.31-2.22(m，1H)，1.91-1.82(m，3H)，1.54-1.50(m，2H)，1.23(s，25H)，0.87-0.82(t，J＝6.6Hz，3H).
Embodiment 79 preparation Pro-Ala-Gln-OCH 2(CH 2) 12CH 3(4r)
Method 355mg (0.58mmol) Boc-Pro-Ala-Gln-OCH according to embodiment 20 2(CH 2) 12-CH 3Make the target compound of 300mg (94%) present embodiment, be the colorless solid powder.Mp：159-160℃；[α] D 25＝-70.1(c＝0.50，CH 3OH)；ESI-MS(m/e)：511[M+H] +；IR(KBr)：3419，2924，2848，2250，1737，1665，1545，1455，1218，1026，711cm -1. 1H-NMR(300MHz，DMSO-d 6)：δ/ppm＝4.38-4.31(m，1H)，4.21-4.15(m，2H)，4.05-3.94(m，2H)，3.89(s，2H)，3.22-3.16(m，2H)，2.33-2.26(m，1H)，2.17-2.12(t，J＝7.8Hz，2H)，1.96-1.75(m，4H)，1.55-1.51(m，2H)，1.23(s，25H)，0.87-0.82(t，J＝6.6Hz，3H).
The thrombus dissolving activity of experimental example 1 4a-r
(6ml/kg i.p.) anaesthetizes with 20% urethane solution with the 200-220g male SD rat.The anesthetized rat dorsal position is fixed, and separates RCCA, in proximal part folder bulldog clamp; Proximal part and distal end penetrate surgical thread respectively, the surgical thread of distal end are clamped with mosquito forceps in fur, in the distal end intubate; Unclamp bulldog clamp, emit about 1ml arterial blood and be contained in the EP pipe of 1ml.The Glass tubing of past vertical fixing (long 15mm, internal diameter 2.5mm, external diameter 5.0mm, the pipe end, seal with plug) and the middle 0.1ml of injection rat artery blood, the rapid thrombus standing bolt that inserts a stainless steel material in past the pipe.This thrombus fixedly spiral uses the Stainless Steel Wire coiled of diameter as 0.2mm, and the long 12mm of spiral part contains 15 bung flanges, and the diameter of bung flange is 1.0mm, and the holder handle links to each other with spiral, and long 7.0mm is the question mark type.Behind the blood coagulation 15min, open the plug of Glass tubing bottom, with the fixing fixing holder handle of spiral of thrombus of tweezers, the thrombus that taking-up is wrapped up by thrombus from Glass tubing is spiral fixedly, accurately weighs.
The bypass intubate constitutes by 3 sections, and the stage casing is a polyethylene rubber tube, long 60mn, internal diameter 3.5mm; Two ends are identical polyethylene tube, long 100mm, internal diameter 1mm; External diameter 2mm, an end of this pipe pull into point pipe (being used to insert rat carotid artery or vein), external diameter 1mm; The outer cover one segment length 7mm of the other end, the polyethylene tube of external diameter 3.5mm (overstriking is used to insert in the polyethylene rubber tube in stage casing).The equal silylanization of the inwall of 3 sections pipes.With the thrombus of thrombus parcel fixedly spiral put into the stage casing polyethylene rubber tube, the two ends of sebific duct are nested with two poly butt ends that add respectively.It is subsequent use to fill with heparin-saline solution (50IU/kg) in will managing through sharp pipe end with syringe.
The left external jugular vein of isolated from rat; Proximal part and distal end penetrate surgical thread respectively; On the left external jugular vein that exposes, cut an angle carefully; The point pipe of the bypass duct that above-prepared is good inserts the proximal part of left external jugular vein opening by angle, simultaneously away from the fixing holder handle of spiral of the interior thrombus in bypass tube stage casing (containing fixedly spiral of the thrombus of accurately weighing).Push the heparin-saline (50IU/kg) of accurate amount with syringe through the point pipe of the other end, this moment, syringe was not withdrawn polyethylene tube, clamped the flexible pipe between syringe and the polyethylene tube with mosquito forceps.Proximal part at RCCA stops blooding with bulldog clamp, RCCA is being cut an angle carefully nearby from bulldog clamp.Extract syringe from the tip of polyethylene tube, the tip of polyethylene tube is inserted the proximal part of artery angle.The two ends of bypass duct all use 4 trumpeter's art sutures and arteriovenous to fix.
With scalp acupuncture with saline water (3ml/kg); Thrust away from the fixing nearly vein place of spiral of thrombus in the stage casing of the physiological salt soln of physiological salt soln of urokinase (20000IU/kg) or different concns compound through bypass tube (containing fixedly spiral of the thrombus of accurately weighing), opens bulldog clamp; Make blood flow flow to vein from artery through bypass duct; This is a rat arteriovenous shut Thrombolysis Model, slowly the liquid in the syringe is injected into (about 6min) in the blood, makes saline water (blank); Urokinase (positive control) or compound of the present invention are through blood circulation, and the sequential action of pressing vein-heart-artery is to thrombus.Timing during from start injection, behind the 1h from bypass duct the fixing spiral of removal of thromboses, accurately weigh.Calculate fixedly of poor quality before and after the spiral administration of thrombus in every rat bypass duct, thrombolysis activity in the body of statistics and assessing compound.The result shows that 4a-r has thrombolysis activity (table 1) in the outstanding body.
Table 1 4a-r is to the influence of rat suppository loss of weight a
Figure BDA0000064453640000211
Figure BDA0000064453640000221
A) n=10, urokinase dosage are 20000IU/kg, and 4a-r dosage is 0.1nmol/kg; B) with the saline water group than p＜0.01.
The thrombus dissolving dose-effect relationship of experimental example 2 4n intravenous administrations
With the method for experimental example 1, choose the best 4n of thrombolysis effect and investigate the thrombolysis activity under 0.1nmol/kg, 0.01nmol/kg, three kinds of dosage of 0.001nmol/kg.The result shows, thrombolytic effect show dose dependency (table 2) in the body of 4n.
The dosage of table 2 4n is to the influence of rat suppository loss of weight a
Figure BDA0000064453640000222
A) n=10; B) organize than p＜0.01 with saline water, 0.01nmol/kg and 0.001nmol/kg; C) organize than p＜0.01 with saline water and 0.001nmol/kg; D) with the saline water group than p＜0.01.
The external thrombus dissolving activity of experimental example 3 4a-r
1) making of thrombosis device
With internal diameter 4mm, external diameter 5.5mm, one section Glass tubing of length 18mm is placed on the quick detachable base of plastics, and the seam crossing of Glass tubing and plastic feet seals with one section emulsion tube.Place a Stainless Steel Wire spiral, screw diameter 1mm, length 20mm in the Glass tubing; The long hook of 2mm that comprises an end; Blood promptly be set in the stainless steel spiral around, can thrombus be hung up when weighing, when hatching, can thrombus be hung in the solution of reaction flask; Do not run into wall, in order to avoid the damage thrombus.
2) making of reaction flask
10ml cillin bottle with the band rubber plug; On rubber plug, wear a Stainless Steel Wire, the end in bottle curves hook, and thrombus hangs on the hook; Be suspended in bottle interior testing compound solution; Stainless Steel Wire can move up and down on rubber plug, regulates the height of thrombus in solution, and it just is immersed in the solution to be measured.The simulation of internal milieu: estimate that according to the rat mean body weight every rat has 13ml blood, if rat thrombus in vivo model, blood that maybe about 8ml can touch thrombus, so adding 8ml solution to be measured in the reaction flask is hatched at 37 ℃ of constant temperature shaking tables.
3) preparation of thrombus
(6ml/kg i.p.), anaesthetizes with 20% urethane with the 350-400g male SD rat; It is fixing to lie on the back, and separates RCCA, and the bulldog clamp folder closes proximal part; The long polyethylene tube of 30mm is inserted in the bulldog clamp top, emits about 3-4ml blood at every turn, approximately can put 2-3 time; 5ml syringe with silylanization injects the Glass tubing that the preparation thrombus is used with the blood of emitting immediately one by one, the stainless steel spiral is put at once.Leave standstill 40min and make thrombosis, afterwards Glass tubing is carefully taken off from base, with fine needle with separating with the Glass tubing inwall around the thrombus; Removal of thromboses hangs on the rubber plug of reaction flask; Add 8ml zero(ppm) water in the reaction flask, thrombus is suspended on leaves standstill 1 hour in the water, remove thrombus surface floating blood.After 1 hour, inhale the moisture on the bolt surface of dehematizing, accurately weigh one by one with filter paper.
4) measure the external thrombolysis activity of 4a-r:
In each reaction flask, refill the physiological salt soln of 4a-r (1nM), as blank, urokinase (100IU/ml) hangs on thrombus in the solution of testing compound as positive control again with saline water, and 37 ℃ of constant temperature shaking table 70rpm were hatched 2 hours.After hatching end, draw surface water with filter paper and accurately weigh one by one again, calculate thrombus at the weight difference that adds solution to be measured front and back, the external thrombolysis activity of statistical appraisal compound.The result shows that 4a-r has outstanding external thrombolysis activity (table 3).
The external thrombolysis activity of table 3 4a-r a
Figure BDA0000064453640000231
A) n=6, urokinase concentration is 100IU/mL, 4a-r concentration is 1nM; B) with the saline water group than P＜0.01; C) with the saline water group than P＜0.05; D) with the saline water group than P＞0.05.
Experimental example 4 concentration are to the influence of the external thrombus dissolving activity of 4h
According to the experimental technique of experimental example 3, choose thrombolysis effect 4h investigation preferably 1nM, the thrombolysis activity under three concentration of 0.1nM and 0.01nM.The result shows, the external thrombolytic effect display density dependency (table 4) of 4h.
The concentration of table 4 4h is to the influence of external thrombolysis activity a
A) n=6; B) with saline water, 0.1nM 4h and 0.01nM 4h than p＜0.01; C) with saline water and 0.01nM 4h than p＜0.01; D) with the saline water group than p＜0.01.
The nanostructure of experimental example 5 4a-r
1) particle diameter of 4a-r nanometer ball in the aqueous solution
On Nano-ZS90 nano particle size determinator, measure 4a-r 10 -5The particle diameter of M.The result shows that 4a-r can be assembled into nanometer ball in the aqueous solution, removes 4c, and outside the 4q, all the other particle diameters are all between 122 to 526nm (table 5).
The particle diameter of the nanometer ball that table 5 4a-r assembles in the aqueous solution
Figure BDA0000064453640000242
2) form of the nanometer ball of 4a-r
It is 1 * 10 that 4a-r is made into concentration -13The aqueous solution of mg/ml, then with this drips of solution on copper mesh, under the JEM-1230 transmission electron microscope, observe the form of nanometer ball behind the dried solvent that volatilizees.Mensuration shows, the nanometer ball of 4a-r formation rule.The transmission electron microscope photo of 4k is described with Fig. 2 as representative.

Claims (3)

1. the general formula that has thrombus dissolving activity is the compound of I:
Pro-Ala-AA-OCH 2(CH 2) 12CH 3General formula I
In the formula, AA be selected from glycine residue, L-Xie Ansuan residue, L-tryptophan residue, L-leucine residue, L-alanine residue, L-methionine residue, L-tyrosine residues, altheine residue, L-Isoleucine residue, L-phenylalanine residue, L-proline residue, L-serine residue, L-threonine residues, L-glutaminate residue, L-asparagicacid residue, L-glutaminic acid residue, L-arginine residues or L-lysine residue one of them.
2. prepare the method for the thrombus dissolving compound of claim 1 described general formula I representative, it is characterized in that the preparation method may further comprise the steps:
1) Boc-Pro is Boc-Pro-Ala with the L-Ala condensation in anhydrous tetrahydro furan in the presence of NSC 57182 and N-hydroxy-succinamide;
2) Boc-AA is Boc-AA-OCH with the condensation of carbon tetradecyl alcohol in anhydrous tetrahydro furan in the presence of dicyclohexyl carbonyl diimine and N-hydroxy benzo triazole 2(CH 2) 12CH 3
3) Boc-AA-OCH in hydrogenchloride-ethyl acetate solution 2(CH 2) 12CH 3Slough Boc and generate AA-OCH 2(CH 2) 12CH 3
4) in the presence of dicyclohexyl carbonyl diimine and N-hydroxy benzo triazole Boc-Pro-Ala in anhydrous tetrahydro furan with AA-OCH 2(CH 2) 12CH 3Condensation is Boc-Pro-Ala-AA-OCH 2(CH 2) 12CH 3
5) Boc-Pro-Ala-AA-OCH in hydrogenchloride-ethyl acetate solution 2(CH 2) 12CH 3Remove Boc and generate Pro-Ala-AA-OCH 2(CH 2) 12CH 3, promptly get.
3. the described compound of claim 1 is in the purposes of preparation in the thrombolytic agent.
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