CN102153623B - Compound with thrombolytic activity and preparation method and application thereof - Google Patents
Compound with thrombolytic activity and preparation method and application thereof Download PDFInfo
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- CN102153623B CN102153623B CN2011100046073A CN201110004607A CN102153623B CN 102153623 B CN102153623 B CN 102153623B CN 2011100046073 A CN2011100046073 A CN 2011100046073A CN 201110004607 A CN201110004607 A CN 201110004607A CN 102153623 B CN102153623 B CN 102153623B
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 97
- 238000002360 preparation method Methods 0.000 title claims abstract description 81
- 230000002537 thrombolytic effect Effects 0.000 title abstract description 6
- COLNVLDHVKWLRT-QMMMGPOBSA-N phenylalanine group Chemical group N[C@@H](CC1=CC=CC=C1)C(=O)O COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims abstract description 17
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 claims abstract description 8
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 claims abstract description 4
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims abstract description 3
- 239000003146 anticoagulant agent Substances 0.000 claims abstract 2
- 239000001257 hydrogen Substances 0.000 claims description 84
- 229910052739 hydrogen Inorganic materials 0.000 claims description 84
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 17
- -1 benzyl ester Chemical class 0.000 claims description 12
- 230000008878 coupling Effects 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 6
- 238000005859 coupling reaction Methods 0.000 claims description 6
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- 238000000034 method Methods 0.000 claims description 5
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 4
- 108010073324 Glutaminase Chemical group 0.000 claims description 2
- 102000009127 Glutaminase Human genes 0.000 claims description 2
- 238000007327 hydrogenolysis reaction Methods 0.000 claims description 2
- 239000007791 liquid phase Substances 0.000 claims description 2
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims 2
- 230000002785 anti-thrombosis Effects 0.000 claims 1
- TYQYRKDGHAPZRF-INIZCTEOSA-N benzyl (2s)-2-amino-3-(1h-indol-3-yl)propanoate Chemical compound O=C([C@H](CC=1C2=CC=CC=C2NC=1)N)OCC1=CC=CC=C1 TYQYRKDGHAPZRF-INIZCTEOSA-N 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 4
- 238000010171 animal model Methods 0.000 abstract description 2
- 125000000405 phenylalanyl group Chemical group 0.000 abstract description 2
- 229960004676 antithrombotic agent Drugs 0.000 abstract 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 abstract 1
- 125000005454 tryptophanyl group Chemical group 0.000 abstract 1
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- 238000006243 chemical reaction Methods 0.000 description 87
- 238000001914 filtration Methods 0.000 description 75
- 238000004809 thin layer chromatography Methods 0.000 description 73
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- 239000007787 solid Substances 0.000 description 50
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- 150000002431 hydrogen Chemical class 0.000 description 43
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- 208000007536 Thrombosis Diseases 0.000 description 27
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 27
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 26
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 26
- 238000001035 drying Methods 0.000 description 26
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- AWJUIBRHMBBTKR-UHFFFAOYSA-N isoquinoline Chemical group C1=NC=CC2=CC=CC=C21 AWJUIBRHMBBTKR-UHFFFAOYSA-N 0.000 description 6
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- NFVNYBJCJGKVQK-ZDUSSCGKSA-N N-[(Tert-butoxy)carbonyl]-L-tryptophan Chemical compound C1=CC=C2C(C[C@H](NC(=O)OC(C)(C)C)C(O)=O)=CNC2=C1 NFVNYBJCJGKVQK-ZDUSSCGKSA-N 0.000 description 3
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
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- OZSSOVRIEPAIMP-ZETCQYMHSA-N (2s)-5-[amino(nitramido)methylidene]azaniumyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanoate Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CCCN=C(N)N[N+]([O-])=O OZSSOVRIEPAIMP-ZETCQYMHSA-N 0.000 description 1
- VVNYDCGZZSTUBC-LURJTMIESA-N (2s)-5-amino-2-[(2-methylpropan-2-yl)oxycarbonylamino]-5-oxopentanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CCC(N)=O VVNYDCGZZSTUBC-LURJTMIESA-N 0.000 description 1
- VRPJIFMKZZEXLR-UHFFFAOYSA-N 2-[(2-methylpropan-2-yl)oxycarbonylamino]acetic acid Chemical compound CC(C)(C)OC(=O)NCC(O)=O VRPJIFMKZZEXLR-UHFFFAOYSA-N 0.000 description 1
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- 108010049003 Fibrinogen Proteins 0.000 description 1
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- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 description 1
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- WUESWDIHTKHGQA-UHFFFAOYSA-N cyclohexylurea Chemical compound NC(=O)NC1CCCCC1 WUESWDIHTKHGQA-UHFFFAOYSA-N 0.000 description 1
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a compound with thrombolytic activity and further discloses the preparation method of the compound and the application of the compound as the anti-thrombotic agent. The general formula of the compound is as follows: H-R1-AA1-Arg-Pro-Ala-Lys-OH or H-AA1-Arg-Pro-Ala-Lys-R2-OH, wherein R1 can be a tryptophanyl, a phenylalanyl or a 1,3,4-trihydroisoquinoline-3-S-formyl, R2 can be a tryptophan residue, a phenylalanine residue or a 1,3,4-trihydroisoquinoline-3-S-carboxylic acid-2-yl, and AA1 can be an alanine residue, a glycine residues or a glutamine residue. The animal model tests confirm that the compound with the general formula has good thrombolytic activity.
Description
Technical field
The present invention relates to compound, relate in particular to compound with thrombus dissolving activity, its preparation method and they, belong to biomedicine field as the application of thrombolytic agent.
Background technology
Vessel embolism, for example myocardial infarction and apoplexy are the highest diseases of mortality ratio in the cardiovascular and cerebrovascular diseases.Seeking thrombolytic agent safely and effectively, is one of focus of cardiovascular and cerebrovascular diseases drug research.(ARPAK is first guide structure to the contriver, has obtained a series of have clear and definite vasoactive oligopeptides (Peng Shiqi, Zhao Ming through structural modification with fibrin degradation product (FDP) P6A; Wang Chao, the pivot Tang Dynasty, Wang Yinye; Cardiovascular active polypeptide and their the synthetic and application in medical science; No. the 57116th, invention patent certificate, Intellectual Property Right Bureau of the RPC, July 28 calendar year 2001); The contriver is through the metabolism research of P6A in the mouse body and analogue, confirmed their meta-bolites and as application prospect (Peng Shiqi, the Zhao Ming of thrombolytic agent; Wang Chao; Xu Youxuan, Wu Yanfen, the meta-bolites of P6A and the application in medical science thereof; Application for a patent for invention number No. 01136780.6, Intellectual Property Right Bureau of the RPC); The contriver intends peptide with N end and the preparation of C end that carboline carboxylate is introduced ARPAK, GRPAK and QRPAK; Make the plan peptide that makes comprise the thrombolysis activity of former peptide sequence, comprised the platelet aggregation inhibitory activity of carboline carboxylate, and have ultraviolet detectability (Yanfen Wu; Ming Zhao; ChaoWang and Shiqi Peng*, Synthesis and Thrombolytic Activity of Pseudopeptides Relatedto Fibrinogen Fragment, Bioorganic&Medicinal Chemistry Letters 12; 2331-2333,2002).Said ultraviolet detectability makes the pharmacokinetic of this comparison peptide become simple and direct.Investigate the heterocyclic structure, the contriver has recognized the ingenious association that Fig. 1 describes.The reasonable popularization of this structure connection is Tetrahydroisoquinoli-dicarboxylic acid moiety, tryptophane and phenylalanine(Phe) to be introduced N end and the C end preparation modified peptides of ARPAK, GRPAK and QRPAK respectively.
Summary of the invention
One of the object of the invention is that ARPAK, GRPAK and three kinds of little peptides of thrombus dissolving of QRPAK are modified, and makes its thrombus dissolving activity better and have an advantage easy to detect.
One of the object of the invention is realized by the following technical programs:
Compound with thrombus dissolving activity, shown in formula (I) or general formula (II):
H-R
1-AA
1-Arg-Pro-Ala-Lys-OH general formula (I);
H-AA
1-Arg-Pro-Ala-Lys-R
2-OH general formula (II).
Wherein, R
1Be selected from tryptophyl, phenylalanyl or 1,3,4-three hydrogen isoquinolines-3-S-formyl radical; R
2Be selected from tryptophan residue, phenylalanine residue or 1,3,4-three hydrogen isoquinolines-3-S-carboxylic acid-2-base; AA
1Be selected from alanine residue (Ala), glycine residue (Gly) or glutaminase residue (Gln).
Two of the object of the invention provides a kind of above-mentioned method with general formula (I) and general formula (II) compound of thrombus dissolving activity for preparing.
Two of the object of the invention is realized through following technical scheme:
A kind ofly prepare above-mentioned method, comprising with general formula (I) or general formula (II) compound of thrombus dissolving activity:
Progressively connect peptide through liquid phase method, prepare the small peptide of following 6 kinds of C end dissociatives or N end dissociative:
(1)Boc-Ala-Arg(NO
2)-Pro-Ala-Lys(Z)-OH，
(2)Boc-Gln-Arg(NO
2)-Pro-Ala-Lys(Z)-OH，
(3)Boc-Gly-Arg(NO
2)-Pro-Ala-Lys(Z)-OH，
(4)H-Gly-Arg(NO
2)-Pro-Ala-Lys(Z)-OBzl，
(5)H-Ala-Arg(NO
2)-Pro-Ala-Lys(Z)-OBzl，
(6) H-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl; 6 kinds of small peptides of Tetrahydroisoquinoli-dicarboxylic acid moiety, tryptophane and phenylalanine(Phe) and above-mentioned C end dissociative or N end dissociative are carried out C end or the coupling of N end, the coupling peptide of preparation protection respectively; The coupling peptide of prepared protection is taken off Boc hydrogenolysis afterwards, obtain target compound.Animal model test confirms, general formula compound of the present invention (I) or (II) have a good thrombus dissolving activity.
Description of drawings
The structural relationship of Fig. 1 quinoline ring and tetrahydroisoquinoline ring, indole ring and phenyl ring.
The synthetic route chart of Fig. 2 The compounds of this invention.
In 1A-C and 2A-C, AA=Ala, Gly, Gln; At 1a, 1 ' a, 1 " a, 1b, 1 ' b and 1 " AA=Ala among the b, R=Phe; At 2a, 2 ' a, 2 " a, 2b, 2 ' b and 2 " AA=Gly among the b, R=Phe; At 3a, 3 ' a, 3 " a, 3b, 3 ' b and 3 " AA=Gln among the b, R=Phe; At 4a, 4 ' a, 4 " a, 4b, 4 ' b and 4 " AA=Ala among the b, R=Trp; At 5a, 5 ' a, 5 " a, 5b, 5 ' b and 5 " b AA=Gly, R=Trp; At 6a, 6 ' a, 6 " a, 6b, 6 ' b and 6 " AA=Gln among the b, R=Trp; At 7a, 7 ' a, 7 " a, 7b, 7 ' b and 7 " AA=Ala among the b, R=isoquinoline 99.9 acyl group; At 8a, 8 ' a, 8 " a, 8b, 8 ' b and 8 " AA=Gly among the b, R=isoquinoline 99.9 acyl group; At 9a, 9 ' a, 9 " a, 9b, 9 ' b and 9 " AA=Gln among the b, R=isoquinoline 99.9 acyl group.
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.
The term that is occurred among the present invention or the explanation of shortenings:
The THF THF
DCC cyclohexyl imide
DCU cyclohexyl urea
The OBzl benzyloxy
The Boc tertbutyloxycarbonyl
HOBt N-hydroxybenzotriazole
The TLC thin-layer chromatography
The DMF N
Embodiment
473mg (2.5mmol) Boc-Ala-OH is dissolved in 10ml anhydrous tetrahydro furan (THF), adds the anhydrous THF solution of 10ml 338mg (2.5mmol) N-hydroxybenzotriazole (HOBt) and 619mg (3mmol) dicyclohexyl carbonyl diimine (DCC) under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution.936mg (2.3mmol) HClLys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine are mixed with the anhydrous THF of 6ml earlier, mix with top active ester solution then.Reaction mixture stirring at room 24h, TLC (CHCl
3: MeOH=30: 1) show that HClLys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue is used acetic acid ethyl dissolution, the filtering insolubles.Filtrating is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.204g (97%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrating, is colorless solid.Mp?88～90℃，
ESI-MS(m/e)543[M+H]
+。
Embodiment 2 preparation HClAla-Lys (Z)-OBzl
With 1.354g (2.5mmol) Boc-Ala-Lys (Z)-OBzl mix with 15ml hydrogenchloride-acetic acid ethyl fluid (4N), stirring at room 3h, TLC (CHCl
3: MeOH=20: 1) show that Boc-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, the title compound that obtains, ESI-MS (m/e) 443 [M+H]
+
Embodiment 3 preparation Boc-Pro-Ala-Lys (Z)-OBzl
538mg (2.5mmol) Boc-Pro-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution of 10ml 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution.1.099g (2.3mmol) HClAla-Lys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine are mixed with the anhydrous THF of 6ml earlier, mix with top active ester solution then.Reaction mixture stirring at room 24h, TLC (CHCl
3: MeOH=20: 1) show that HClAla-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue is used acetic acid ethyl dissolution, the filtering insolubles.Filtrating is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 2.847g (98%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrating.Be colorless solid.Mp?82～83.4℃，
ESI-MS(m/e)640[M+H]
+。
Embodiment 4 preparation HClPro-Ala-Lys (Z)-OBzl
With the mixing of 1.596g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N), stirring at room 3h, TLC (CHCl
3: MeOH=20: 1) show that Boc-Pro-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, the title compound that obtains, ESI-MS (m/e) 640 [M+H]
+
Embodiment 5 preparation Boc-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 798mg (2.5mmol) Boc-Arg (NO
2)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution of 10ml 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution.1.322g (2.3mmol) HClPro-Ala-Lys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine are mixed with the anhydrous THF of 6ml earlier, mix with top active ester solution again.Reaction mixture stirring at room 24h, TLC (CHCl
3: MeOH=20: 1) show that HClPro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue is used acetic acid ethyl dissolution, the filtering insolubles.Filtrating is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.642g (85%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrating.Be colorless solid.Mp?83～85℃，
ESI-MS(m/e)842[M+H]
+。
Embodiment 6 preparation HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 2.099g (2.5mmol) Boc-Arg (NO
2The mixing of)-Pro-Ala-Lys (Z)-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N), stirring at room 3h, TLC (CHCl
3: MeOH=20: 1) show Boc-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, the title compound that obtains, ESI-MS (m/e) 742 [M+H]
+
Embodiment 7 preparation Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
473mg (2.5mmol) Boc-Ala-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution of 10ml338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution.With 1.785g (2.3mmol) HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine mix with the anhydrous THF of 6ml earlier, mix with top active ester solution then.Reaction mixture room temperature reaction 24h, TLC (CHCl
3: MeOH=20: 1) show HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue is used acetic acid ethyl dissolution, the filtering insolubles.Filtrating is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.802g (86%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrating.Be colorless solid.Mp?87～89℃，ESI-MS(m/e)912[M+H]
+。
Embodiment 8 preparation HClAla-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 2.277g (2.5mmol) Boc-Ala-Arg (NO
2The mixing of)-Pro-Ala-Lys (Z)-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N), stirring at room 3h, TLC (CHCl
3: MeOH=20: 1) show Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, the title compound that obtains.ESI-MS(m/e)812[M+H]
+。
With 2.277g (2.5mmol) Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl is dissolved in the 10ml methyl alcohol, and 0 ℃ of NaOH aqueous solution that adds 1.5mol/l is down regulated pH value to 11, stirring reaction 0.5hr, and the TLC detection reaction finishes.Add saturated KHSO
4Regulate pH value to 6～7, the salt that filtering is separated out, whole methyl alcohol are removed in the filtrate decompression distillation, and residuary water solution continues to use KHSO
4Regulate pH value to 2, with ethyl acetate extraction three times, ethyl acetate layer merges, give a baby a bath on the third day after its birth time with saturated NaCl collection, and dry 3 hours of anhydrous Na SO4, filtration, filtrate decompression is concentrated removes ETHYLE ACETATE, obtains 1.973g (96%) target compound.ESI-MS(m/e)822[M+H]
+。
Embodiment 10 preparation Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
438mg (2.5mmol) Boc-Gly-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution of 10ml 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution.With 1.785g (2.3mmol) HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine mix with the anhydrous THF of 6ml earlier, mix with top active ester solution again.Reaction mixture stirring at room 24h, TLC (CHCl
3: MeOH=20: 1) show HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue is used acetic acid ethyl dissolution, the filtering insolubles.Filtrating is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.857g (90%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrating.Be colorless solid.Mp?85～87℃，ESI-MS(m/e)898[M+H]
+。
Embodiment 11 preparation HClGly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 2.242g (2.5mmol) Boc-Gly-Arg (NO
2The mixing of)-Pro-Ala-Lys (Z)-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N), stirring at room 3h, TLC (CHCl
3: MeOH=20: 1) show Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, the title compound that obtains, ESI-MS (m/e) 798 [M+H]
+
Embodiment 12 preparation Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OH
With 2.242g (2.5mmol) Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl l is dissolved in the 10ml methyl alcohol, and 0 ℃ of NaOH aqueous solution that adds 1.5mol/l is down regulated pH value to 11, stirring reaction 0.5hr, and the TLC detection reaction finishes.Add saturated KHSO
4Regulate pH value to 6～7, the salt that filtering is separated out, whole methyl alcohol are removed in the filtrate decompression distillation, and residuary water solution continues to use KHSO
4Regulate pH value to 2, with ethyl acetate extraction three times, ethyl acetate layer merges, give a baby a bath on the third day after its birth time with saturated NaCl collection, and dry 3 hours of anhydrous Na SO4, filtration, filtrate decompression is concentrated removes ETHYLE ACETATE, obtains 1.957g (97%) target compound.ESI-MS(m/e)808[M+H]
+。
Embodiment 13 preparation Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
615mg (2.5mmol) Boc-Gln-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution of 10ml 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution.With 1.785g (2.3mmol) HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine mix with the anhydrous THF of 6ml earlier, mix with top active ester solution again.Reaction mixture stirring at room 24h, TLC (CHCl
3: MeOH=10: 1) show HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue is used acetic acid ethyl dissolution, the filtering insolubles.Filtrating is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.936g (80%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrating.Be colorless solid.Mp?91～92℃，ESI-MS(m/e)969[M+H]
+。
Embodiment 14 preparation HClGln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 2.420g (2.5mmol) Boc-Gln-Arg (NO
2The mixing of)-Pro-Ala-Lys (Z)-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N), stirring at room 3h, TLC (CHCl
3: MeOH=20: 1) show Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, and the title compound that obtains is used for step reaction down.ESI-MS(m/e)869[M+H]
+。
Embodiment 15 preparation Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OH
With 2.420g (2.5mmol) Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl is dissolved in the 10ml methyl alcohol, and 0 ℃ of NaOH aqueous solution that adds 1.5mol/l is down regulated pH value to 11, stirring reaction 0.5hr, and the TLC detection reaction finishes.Add saturated KHSO
4Regulate pH value to 6～7, the salt that filtering is separated out, whole methyl alcohol are removed in the filtrate decompression distillation, and residuary water solution continues to use KHSO
4Regulate pH value to 2, with ethyl acetate extraction three times, ethyl acetate layer merges, give a baby a bath on the third day after its birth time with saturated NaCl collection, and dry 3 hours of anhydrous Na SO4, filtration, filtrate decompression is concentrated removes ETHYLE ACETATE, obtains 2.088g (95%) target compound.ESI-MS(m/e)879[M+H]
+。
Embodiment 16 preparation Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl
With 821mg (1.0mmol) Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OH is dissolved in the 3ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds that 393mg (1.35mmol) HClPhe-OBzl is dissolved in the 5ml dry DMF inward and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration Boc-Ala-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OH disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 920mg (87%) target compound, is colorless solid.ESI-MS(m/e)1059[M+H]
+。
Embodiment 17 preparation H-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 423mg (0.40mmol) Boc-Ala-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-Phe-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 360mg (94%) title compound, ESI-MS (m/e) 959 [M+H]
+
Embodiment 18 preparation H-Ala-Arg-Pro-Ala-Lys-Phe-OH (1a)
With 398mg (0.4mmol) HClAla-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClAla-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 181mg (66%) title compound, be colorless solid.ESI-MS(m/z)689[M+H]
+。
Embodiment 19 preparation Boc-Phe-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
317mg (1.2mmol) Boc-Phe-OH is dissolved among the anhydrous THF of 3ml, and ice bath adds the solution of 151mg (1.2mmol) HOBt, 268mg (1.3mmol) DCC and the anhydrous THF of 10ml down, and the mixture ice bath stirs 30min down.Add 847mg (1.0mmol) HClAla-Arg (NO inward
2)-Pro-Ala-Lys (Z)-OBzl is dissolved in the 3ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration HClAla-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 868mg (82%) target compound, is colorless solid.ESI-MS(m/e)1059[M+H]
+。
Embodiment 20 preparation H-Phe-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 400mg (0.40mmol) Boc-Phe-Ala-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 372mg (97%) title compound, ESI-MS (m/e) 959 [M+H]
+
Embodiment 21 preparation H-Phe-Ala-Arg-Pro-Ala-Lys-OH (1b)
With 398mg (0.4mmol) HClPhe-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClPhe-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 128mg (47%) title compound, be colorless solid.ESI-MS(m/z)689[M+H]
+。
Embodiment 22 preparation Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl
With 821mg (1.0mmol) Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OH is dissolved in the 3ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds that 446mg (1.35mmol) HClTrp-OBzl is dissolved in the 5ml dry DMF inward and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration Boc-Ala-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OH disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 991mg (89%) target compound, is colorless solid.ESI-MS(m/e)1115[M+H]
+。
Embodiment 23 preparation H-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 446mg (0.40mmol) Boc-Ala-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-Trp-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 390mg (96%) title compound, ESI-MS (m/e) 1015 [M+H]
+
Embodiment 24 preparation H-Ala-Arg-Pro-Ala-Lys-Trp-OH (2a)
With 420mg (0.4mmol) HClAla-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClAla-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 204mg (70%) title compound, be colorless solid.ESI-MS(m/z)728[M+H]
+。[α]
D 20＝-22°(c＝1.4，H
2O)。
Embodiment 25 preparation Boc-Trp-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
364mg (1.2mmol) Boc-Trp-OH is dissolved among the anhydrous THF of 3ml, and ice bath adds the solution of 151mg (1.2mmol) HOBt, 268mg (1.3mmol) DCC and the anhydrous THF of 10ml down, and the mixture ice bath stirs 30min down.Add 847mg (1.0mmol) HClAla-Arg (NO inward
2)-Pro-Ala-Lys (Z)-OBzl is dissolved in the 3ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration HClAla-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 937mg (84%) target compound, is colorless solid.ESI-MS(m/e)1115[M+H]
+。
Embodiment 26 preparation H-Trp-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 446mg (0.40mmol) Boc-Trp-Ala-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Trp-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 386mg (95%) title compound, ESI-MS (m/e) 1015 [M+H]
+
Embodiment 27 preparation H-Trp-Ala-Arg-Pro-Ala-Lys-OH (2b)
With 420mg (0.4mmol) HClTrp-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClTrp-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 198mg (68%) title compound, be colorless solid.ESI-MS(m/z)728[M+H]
+。
Embodiment 28 preparations 1,3,4-three hydrogen isoquinolines-3-S-carboxylic acid
In 4.0g (24.2mmol) L-phenylalanine(Phe), dropwise add 21.6ml formaldehyde earlier, dropwise add the 36ml35% concentrated hydrochloric acid again.The suspension oil bath that obtains is heated to 2 two hours phenylalanine(Phe)s of 80-90 ℃ of stirring and dissolves fully, reacts after 2.5 hours, and beginning adularescent deposition generates, and reacts TLC (CHCl 7 hours
3/ CH
3OH=5/1, RF=0.56) plate shows that raw material disappears, suction filtration gets colorless solid 4.2g; With the gained colorless solid join in the 85.6ml80% ethanol oil bath be heated to 80 ℃ 9 hours, the colorless solid dissolving is cooled to room temperature; Slowly drip 1ml (1.376g Pottasium Hydroxide is dissolved in 1ml zero(ppm) water) potassium hydroxide solution; There is colourless deposition to separate out, crosses and filter 4.02g (94%) title compound, be colorless solid.ESI-MS(m/z)178[M+H]
+。
Embodiment 29 preparation N-Boc-1,3,4-three hydrogen isoquinolines-3-S-carboxylic acid
The following 2.49g of ice bath (62.15mmol) sodium hydroxide is separated the water in 62ml, and earlier with 10.0g (56.5mmol) 1,2,3,4 ,-tetrahydrochysene-isoquinoline 99.9-3-carboxylic acid suspends wherein, adds 40ml THF and 14.8g (67.8mmol) more inward (Boc)
2The solution of O.Reaction mixture stirs 48h, solution becomes clarification, TLC (ethyl acetate/petroleum ether: 1: 3) demonstration 1,2,3,4, and-tetrahydrochysene-isoquinoline 99.9-3-carboxylic acid disappears.Stop reaction, concentrating under reduced pressure, residue and use acetic acid ethyl dissolution.The solution that obtains is used 5%KHSO successively
4The aqueous solution is washed, saturated NaCl wash water solution is washed and washed.Isolating organic layer stirs 24h with anhydrous sodium sulfate drying, filtration, concentrating under reduced pressure, residue and 100ml ethyl acetate/petroleum ether (1/10) mixing solutions, gets 12.50g (80%) title compound, is colorless solid.ESI-MS(m/z)278[M+H]
+。
Embodiment 30 preparations 1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate
Toward 280mg (0.86mmol) Cs
2CO
3With add 304mg (1.71mmol) 1,2,3, the solution of 4-tetrahydrochysene-β-Ka Lin-3-S-carboxylic acid and 4ml absolute ethyl alcohol in the solution of 1ml water.Concentrating under reduced pressure behind the reaction mixture stirring at room 40min generates colorless solid.This colorless solid and 5ml dry DMF are processed solution, in this solution, add 0.2ml (1.75mmol) bromobenzyl.Reaction mixture stirs 16h in 50 ℃.The CsBr deposition that filtering generates, filtrating is used saturated NaCO successively
3The aqueous solution is washed (30ml * 3), the saturated NaCl aqueous solution is washed (30ml * 3), 5%KHSO
4The aqueous solution is washed (30ml * 3).Isolating organic layer gets 367mg (80%) title compound with anhydrous sodium sulfate drying, filtration, concentrating under reduced pressure, is colorless solid.ESI-MS(m/z)268[M+H]
+。
Embodiment 31 preparation N-[Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate
With 821mg (1.0mmol) Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OH is dissolved in the 3ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds 413mg (1.35mmol) 1,3 inward, and 4-three hydrogen-β-Ka Lin-3-S-benzyl carboxylate is dissolved in the 5ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration Boc-Ala-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OH disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 888mg (80%) target compound, is colorless solid.ESI-MS(m/e)1111[M+H]
+。
Embodiment 32 preparation N-[H-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 444mg (0.40mmol) N-[Boc-Ala-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)]-1,2,3,4-tetrahydroisoquinoline-3-S-benzyl carboxylate.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show N-[Boc-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,2,3,4-tetrahydroisoquinoline-3-S-benzyl carboxylate disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 388mg (96%) title compound, ESI-MS (m/e) 1011 [M+H]
+
Embodiment 33 preparation N-(H-Ala-Arg-Pro-Ala-Lys)-1,3,4-three hydrogen isoquinolines-3-S-carboxylic acid (3a)
With 404mg (0.4mmol) N-[H-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows N-[H-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 222mg (75%) title compound, be colorless solid.ESI-MS(m/z)740[M+H]
+。[α]
D 20＝-26°(c＝1.08，H
2O).
Embodiment 34 preparation N-(Boc-1,3,4-three hydrogen isoquinolines-3-S-formyl)-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 360mg (1.3mmol) N-Boc-1,3,4-three hydrogen isoquinolines-3-S-carboxylic acid is dissolved in the 10ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds 810mg (1.0mmol) HClAla-Arg (NO inward
2)-Pro-Ala-Lys (Z)-OBzl is dissolved in the 5ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration HClAla-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 887mg (83%) target compound, is colorless solid.ESI-MS(m/e)1070[M+H]
+。
Embodiment 35 preparation N-(1,3,4-three hydrogen isoquinolines-3-S-formyl)-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide 4 times and add 428mg (0.40mmol) N-(Boc-1,3,4-three hydrogen isoquinolines-3-S-formyl)-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show N-(Boc-1,3,4-three hydrogen isoquinolines-3-S-formyl)-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 364mg (94%) title compound, ESI-MS (m/e) 970 [M+H]
+
Embodiment 36 preparation N-(1,3,4-three hydrogen isoquinolines-3-S-formyl)-Ala-Arg-Pro-Ala-Lys-OH (3b)
With 388mg (0.4mmol) N-(1,3,4-three hydrogen isoquinolines-3-S-formyl)-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows N-(1,3,4-three hydrogen isoquinolines-3-S-formyl)-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 236mg (80%) title compound, be colorless solid.ESI-MS(m/z)740[M+H]
+。[α]
D 20＝-38°(c＝1.5，H
2O).
Embodiment 37 preparation Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl
With 807mg (1.0mmol) Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OH is dissolved in the 3ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds that 393mg (1.35mmol) HClPhe-OBzl is dissolved in the 5ml dry DMF inward and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration Boc-Gly-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OH disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 798mg (89%) target compound, is colorless solid.ESI-MS(m/e)988[M+H]
+。
Embodiment 38 preparation H-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 383mg (0.40mmol) Boc-Gly-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-Phe-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 341mg (96%) title compound, ESI-MS (m/e) 888 [M+H]
+
Embodiment 39 preparation H-Gly-Arg-Pro-Ala-Lys-Phe-OH (4a)
With 369mg (0.4mmol) HClAla-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClGly-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 189mg (70%) title compound, be colorless solid.ESI-MS(m/z)675[M+H]
+。
Embodiment 40 preparation Boc-Phe-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
317mg (1.2mmol) Boc-Phe-OH is dissolved among the anhydrous THF of 3ml, and ice bath adds the solution of 151mg (1.2mmol) HOBt, 268mg (1.3mmol) DCC and the anhydrous THF of 10ml down, and the mixture ice bath stirs 30min down.Add 834mg (1.0mmol) HClGly-Arg (NO inward
2)-Pro-Ala-Lys (Z)-OBzl is dissolved in the 3ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration HClGly-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 888mg (90%) target compound, is colorless solid.ESI-MS(m/e)988[M+H]
+。
Embodiment 41 preparation H-Phe-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 400mg (0.40mmol) Boc-Phe-Gly-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 348mg (98%) title compound, ESI-MS (m/e) 888 [M+H]
+
Embodiment 42 preparation H-Phe-Gly-Arg-Pro-Ala-Lys-OH (4b)
With 355mg (0.4mmol) HClPhe-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClPhe-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 167mg (62%) title compound, be colorless solid.ESI-MS(m/z)675[M+H]
+。[α]
D 20＝-24°
Embodiment 43 preparation Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl
With 808mg (1.0mmol) Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OH is dissolved in the 3ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds that 446mg (1.35mmol) HClTrp-OBzl is dissolved in the 5ml dry DMF inward and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration Boc-Gly-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OH disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 1013mg (92%) target compound, is colorless solid.ESI-MS(m/e)1101[M+H]
+。
Embodiment 44 preparation H-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 444mg (0.40mmol) Boc-Gly-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-Trp-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 380mg (95%) title compound, ESI-MS (m/e) 1001 [M+H]
+
Embodiment 45 preparation H-Gly-Arg-Pro-Ala-Lys-Trp-OH (5a)
With 415mg (0.4mmol) HClGly-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClGly-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 214mg (75%) title compound, be colorless solid.ESI-MS(m/z)714[M+H]
+。[α]
D 20＝-22°(c＝1.4，H
2O)。
Embodiment 46 preparation Boc-Trp-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
364mg (1.2mmol) Boc-Trp-OH is dissolved among the anhydrous THF of 3ml, and ice bath adds the solution of 151mg (1.2mmol) HOBt, 268mg (1.3mmol) DCC and the anhydrous THF of 10ml down, and the mixture ice bath stirs 30min down.Add 834mg (1.0mmol) HClGly-Arg (NO inward
2)-Pro-Ala-Lys (Z)-OBzl is dissolved in the 3ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration HClGly-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 947mg (86%) target compound, is colorless solid.ESI-MS(m/e)1101[M+H]
+。
Embodiment 47 preparation H-Trp-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 440mg (0.40mmol) Boc-Trp-Gly-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Trp-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 384mg (96%) title compound, ESI-MS (m/e) 1001 [M+H]
+
Embodiment 48 preparation H-Trp-Gly-Arg-Pro-Ala-Lys-OH (5b)
With 415mg (0.4mmol) HClTrp-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClTrp-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 200mg (70%) title compound, be colorless solid.ESI-MS(m/z)714[M+H]
+。[α]
D 20＝-24°(c＝1.6，H
2O)。
Embodiment 49 preparation N-[Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate
With 807mg (1.0mmol) Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OH is dissolved in the 3ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds 413mg (1.35mmol) 1,3 inward, and 4-three hydrogen-β-Ka Lin-3-S-benzyl carboxylate is dissolved in the 5ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration Boc-Gly-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OH disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 932mg (85%) target compound, is colorless solid.ESI-MS(m/e)1097[M+H]
+。
Embodiment 50 preparation N-[H-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 438mg (0.40mmol) N-[Boc-Gly-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show N-[Boc-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 387mg (97%) title compound, ESI-MS (m/e) 997 [M+H]
+
Embodiment 51 preparation N-(H-Gly-Arg-Pro-Ala-Lys)-1,3,4-three hydrogen isoquinolines-3-S-carboxylic acid (6a)
With 398mg (0.4mmol) N-[H-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows N-[H-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 227mg (78%) title compound, be colorless solid.ESI-MS(m/z)726[M+H]
+。[α]
D 20＝-16°(c＝1.6，H
2O).
Embodiment 52 preparation N-(Boc-1,3,4-three hydrogen isoquinolines-3-S-formyl)-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 360mg (1.3mmol) N-Boc-1,3,4-three hydrogen isoquinolines-3-S-carboxylic acid is dissolved in the 10ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds 834mg (1.0mmol) HClGly-Arg (NO inward
2)-Pro-Ala-Lys (Z)-OBzl is dissolved in the 5ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration HClGly-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 897mg (85%) target compound, is colorless solid.ESI-MS(m/e)1056[M+H]
+。
Embodiment 53 preparation N-(1,3,4-three hydrogen isoquinolines-3-S-formyl)-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide 4 times and add 422mg (0.40mmol) N-(Boc-1,3,4-three hydrogen isoquinolines-3-S-formyl)-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show N-[Boc-1,3,4-three hydrogen isoquinolines-3-S-formyl]-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 367mg (96%) title compound, ESI-MS (m/e) 956 [M+H]
+
Embodiment 54 preparation N-(1,3,4-three hydrogen isoquinolines-3-S-formyl)-Gly-Arg-Pro-Ala-Lys-OH (6b)
With 382mg (0.4mmol) N-(1,3,4-three hydrogen isoquinolines-3-S-formyl)-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows N-[1,3,4-three hydrogen isoquinolines-3-S-formyl]-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 238mg (82%) title compound, be colorless solid.ESI-MS(m/z)726[M+H]
+。[α]
D 20＝-24°(c＝1.5，H
2O).
Embodiment 55 preparation Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl
With 878mg (1.0mmol) Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OH is dissolved in the 3ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds that 393mg (1.35mmol) HClPhe-OBzl is dissolved in the 5ml dry DMF inward and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration Boc-Gln-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OH disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 948mg (85%) target compound, is colorless solid.ESI-MS(m/e)1116[M+H]
+。
Embodiment 56 preparation H-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 446mg (0.40mmol) Boc-Gln-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-Phe-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 374mg (92%) title compound, ESI-MS (m/e) 1016 [M+H]
+
Embodiment 57 preparation H-Gln-Arg-Pro-Ala-Lys-Phe-OH (7a)
With 406mg (0.4mmol) HClGln-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClGln-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 206mg (69%) title compound, be colorless solid.ESI-MS(m/z)746[M+H]
+。[α]
D 20＝
Embodiment 58 preparation Boc-Phe-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
317mg (1.2mmol) Boc-Phe-OH is dissolved among the anhydrous THF of 3ml, and ice bath adds the solution of 151mg (1.2mmol) HOBt, 268mg (1.3mmol) DCC and the anhydrous THF of 10ml down, and the mixture ice bath stirs 30min down.Add 868mg (1.0mmol) HClGln-Arg (NO inward
2)-Pro-Ala-Lys (Z)-OBzl is dissolved in the 3ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration HClGln-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 915mg (82%) target compound, is colorless solid.ESI-MS(m/e)1116[M+H]
+。
Embodiment 59 preparation H-Phe-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 446mg (0.40mmol) Boc-Phe-Gln-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-Phe-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 390mg (96%) title compound, ESI-MS (m/e) 1016 [M+H]
+
Embodiment 60 preparation H-Phe-Gln-Arg-Pro-Ala-Lys-OH (7b)
With 420mg (0.4mmol) HClPhe-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClPhe-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 140mg (47%) title compound, be colorless solid.ESI-MS(m/z)746[M+H]
+。[α]
D 20＝
Embodiment 61 preparation Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl
With 878mg (1.0mmol) Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OH is dissolved in the 3ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds that 446mg (1.35mmol) HClTrp-OBzl is dissolved in the 5ml dry DMF inward and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration Boc-Gln-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OH disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 1055mg (90%) target compound, is colorless solid.ESI-MS(m/e)1172[M+H]
+。
Embodiment 62 preparation H-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 468mg (0.40mmol) Boc-Gln-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-Trp-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 407mg (95%) title compound, ESI-MS (m/e) 1072 [M+H]
+
Embodiment 63 preparation H-Gln-Arg-Pro-Ala-Lys-Trp-OH (8a)
With 428mg (0.4mmol) HClGln-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClGln-Arg (NO
2)-Pro-Ala-Lys (Z)-Trp-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 236mg (75%) title compound, be colorless solid.ESI-MS(m/z)785[M+H]
+。[α]
D 20＝-50°(c＝1.4，H
2O)。
Embodiment 64 preparation Boc-Trp-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
364mg (1.2mmol) Boc-Trp-OH is dissolved among the anhydrous THF of 3ml, and ice bath adds the solution of 151mg (1.2mmol) HOBt, 268mg (1.3mmol) DCC and the anhydrous THF of 10ml down, and the mixture ice bath stirs 30min down.Add 868mg (1.0mmol) HClGln-Arg (NO inward
2)-Pro-Ala-Lys (Z)-OBzl is dissolved in the 3ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration HClGln-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 1008mg (86%) target compound, is colorless solid.ESI-MS(m/e)1172[M+H]
+。
Embodiment 65 preparation H-Trp-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 468mg (0.40mmol) Boc-Trp-Gln-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show Boc-Trp-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 1028mg (96%) title compound, ESI-MS (m/e) 1072 [M+H]
+
Embodiment 66 preparation H-Trp-Gln-Arg-Pro-Ala-Lys-OH (8b)
With 428mg (0.4mmol) HClTrp-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows HClTrp-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 198mg (68%) title compound, be colorless solid.ESI-MS(m/z)728[M+H]
+。[α]
D 20＝-52°
Embodiment 67 preparation N-[Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate
With 878mg (1.0mmol) Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OH is dissolved in the 3ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds 413mg (1.35mmol) 1,3 inward, and 4-three hydrogen-β-Ka Lin-3-S-benzyl carboxylate is dissolved in the 5ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration Boc-Gln-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OH disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 1248mg (81%) target compound, is colorless solid.ESI-MS(m/e)1168[M+H]
+。
Embodiment 68 preparation N-[H-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide to add 467mg (0.40mmol) N-[Boc-Gln-Arg (NO for 4 times
2)-Pro-Ala-Lys (Z)]-1,2,3,4-tetrahydroisoquinoline-3-S-benzyl carboxylate.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show N-[Boc-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,2,3,4-tetrahydroisoquinoline-3-S-benzyl carboxylate disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 414mg (97%) title compound, ESI-MS (m/e) 1068 [M+H]
+
Embodiment 69 preparation N-(H-Gln-Arg-Pro-Ala-Lys)-1,3,4-three hydrogen isoquinolines-3-S-carboxylic acid (9a)
With 427mg (0.4mmol) N-[H-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows N-[H-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)]-1,3,4-three hydrogen isoquinolines-3-S-benzyl carboxylate disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 245mg (77%) title compound, be colorless solid.ESI-MS(m/z)797[M+H]
+。[α]
D 20＝-64°(c＝1.08，H
2O).
Embodiment 70 preparation N-(Boc-1,3,4-three hydrogen isoquinolines-3-S-formyl)-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 360mg (1.3mmol) N-Boc-1,3,4-three hydrogen isoquinolines-3-S-carboxylic acid is dissolved in the 10ml dry DMF, and ice bath adds the solution of 151mg (1.2mmol) HOBt and 268mg (1.3mmol) DCC and the anhydrous THF of 20ml down.The reaction mixture ice bath stirs down 30min, adds 904mg (1.0mmol) HClGln-Arg (NO inward
2)-Pro-Ala-Lys (Z)-OBzl is dissolved in the 5ml dry DMF and transfers the solution of pH8.5 with N-methylmorpholine.Reaction mixture stirs 2h, stirring at room 20h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) demonstration HClGln-Arg (NO for 0 ℃
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering DCU, concentrating under reduced pressure, residue are with dissolved in chloroform, the residual DCU of elimination again.Chloroform layer successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution washing and washing.The chloroform layer of telling with anhydrous sodium sulfate drying, filtration, filtrate decompression concentrate, residue grinds with ETHYLE ACETATE, obtains 957mg (85%) target compound, is colorless solid.ESI-MS(m/e)1127[M+H]
+。
Embodiment 71 preparation N-(1,3,4-three hydrogen isoquinolines-3-S-formyl)-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
Under the ice bath 10ml anhydrous hydrogen chloride/ethyl acetate solution (6N) is stirred 10min, divide 4 times and add 450mg (0.40mmol) N-(Boc-1,3,4-three hydrogen isoquinolines-3-S-formyl)-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl.Stirring at room 3h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) show N-(Boc-1,3,4-three hydrogen isoquinolines-3-S-formyl)-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction solution is evaporated to dried, residue mix with anhydrous diethyl ether again press be concentrated into dried.This operation repetition 5 times.Last residue grinds with anhydrous diethyl ether, obtains 394mg (96%) title compound, ESI-MS (m/e) 1027 [M+H]
+
Embodiment 72 preparation N-(1,3,4-three hydrogen isoquinolines-3-S-formyl)-Gln-Arg-Pro-Ala-Lys-OH (9b)
With 410mg (0.4mmol) N-(1,3,4-three hydrogen isoquinolines-3-S-formyl)-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl adds 300mgPd/C (5%) after being dissolved in the mixed solution of 9ml ethanol and 6ml water, and the reaction flask decompression exhaust also feeds hydrogen.This operation repetition 5 times.Logical hydrogen of reaction mixture and stirring at room 24h, TLC (chloroform/methanol/Glacial acetic acid min. 99.5,5/1/0.2) shows N-[1,3,4-three hydrogen isoquinolines-3-S-formyl]-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates.Resistates is with C18 column purification (2.5% acetonitrile/1%HAc/H
2O), get 264mg (83%) title compound, be colorless solid.ESI-MS(m/z)797[M+H]
+。[α]
D 20＝-32°(c＝1.5，H
2O).
Test Example 1 The compounds of this invention thrombus dissolving activity determination test
1) rat operation and apparatus
(male, 220～230g) press 1200mgkg to the SD rat
-1Dosage abdominal injection urethane solution is anaesthetized.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, be contained in the 1ml sub warhead.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,, from Glass tubing, take out fixedly spiral of the thrombus that wrapped up by thrombus carefully with the fixing fixing holder handle of spiral of thrombus of tweezers.Accurately weigh.
2) rat neck arteriovenous shut intubate
The bypass intubate constitutes by 3 sections, and the stage casing is a polyethylene rubber tube, long 60.0mm, internal diameter 3.5mm; Two ends are identical polyethylene tube; Pipe range 100.0mm, the end that internal diameter 1.0mm, external diameter 2.0mm should manage pull into point pipe (being used to insert rat carotid artery or vein); External diameter is 1.0mm; The outer cover one segment length 7.0mm of the other end, external diameter are the equal silylanization of inwall of 3 sections pipes of polyethylene tube (overstriking is used to insert in the polyethylene rubber tube in stage casing) of 3.5mm.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.Fill with heparin-saline solution (50IUkg in will managing through sharp pipe end with syringe
-1), subsequent use.
Continue the tracheae of dissociation anesthesia rat, and do trachea cannula.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 (50IUkg of accurate amount through the point pipe of the other end with syringe
-1), 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.
3) administration
(dosage is 3mlkg with saline water with scalp acupuncture
-1), physiological salt soln (dosage is 20000IU/kg) or 18 kinds of compound (1a-9a of the present invention of urokinase; Thrust away from the fixing nearly vein place of spiral of thrombus in the stage casing of physiological salt soln 1b-9b) (dosage is 10 μ mol/kg) through bypass tube (containing fixedly spiral of the thrombus of accurately weighing); Open bulldog clamp, make blood flow flow to vein through bypass duct from artery, this is a rat arteriovenous shut Thrombolysis Model; Slowly the liquid in the syringe is injected in the blood; Make saline water (blank), urokinase (positive control) or linear peptides (therapeutical agent) 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.Ask fixedly of poor quality before and after the spiral administration of thrombus in every rat bypass duct; The thrombus quality difference (
) of each group of statistics, and do the t check.
4) result
Thrombus quality difference is listed table 1 in.The data declaration of table 1, (1a-9a 1b-9b) all has outstanding thrombus dissolving activity to 18 kinds of compounds of the present invention under 10 μ mol/kg dosage.Under 10 μ mol/kg dosage except that the specific activity 20000IU/kg dosage UK of 1a, 1b, 2b, 8a, a 8b and 9b6 compound active weak, all the other 12 compounds active all suitable with UK.It is thus clear that compound of the present invention is outstanding thrombolytic agent.
Table 1. The compounds of this invention (1a-9a, thrombus dissolving activity test-results 1b-9b)
N=10,1a, b-9a, the dosage of b are 10 μ mol/kg, the dosage of UK is 20000IU/kg; A) with NS ratio, P＜0.001; B) with NS ratio, P＜0.01; C) with UK ratio, P＞0.05.
Claims (3)
1. the compound shown in the general formula (II):
H-AA
1-Arg-Pro-Ala-Lys-R
2-OH general formula (II);
Wherein, R
2Be selected from tryptophan residue, phenylalanine residue or 1,3,4-three hydrogen isoquinolines-3-S-carboxylic acid-2-base; AA
1Be selected from alanine residue, glycine residue or glutaminase residue.
2. prepare the method for the compound of claim 1, comprising:
Progressively connect peptide through liquid phase method, prepare following 3 kinds of small peptides:
(1)Boc-Ala-Arg(NO
2)-Pro-Ala-Lys(Z)-OH，
(2)Boc-Gln-Arg(NO
2)-Pro-Ala-Lys(Z)-OH，
(3)Boc-Gly-Arg(NO
2)-Pro-Ala-Lys(Z)-OH，
With 1,3,3 kinds of small peptides of 4-three hydrogen isoquinolines-3-S-benzyl carboxylate, tryptophan benzyl ester or phenylalanine(Phe) benzyl ester and above-mentioned C end dissociative carry out the coupling of C end, the coupling peptide of preparation protection respectively; Hydrogenolysis promptly got to slough the protection base after the coupling peptide of prepared protection taken off tertbutyloxycarbonyl.
3. the compound of claim 1 is in the purposes of preparation in the antithrombotic reagent.
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