CN102485744B - 溶血栓的寡肽化合物及其制备方法和应用 - Google Patents
溶血栓的寡肽化合物及其制备方法和应用 Download PDFInfo
- Publication number
- CN102485744B CN102485744B CN201010576378.8A CN201010576378A CN102485744B CN 102485744 B CN102485744 B CN 102485744B CN 201010576378 A CN201010576378 A CN 201010576378A CN 102485744 B CN102485744 B CN 102485744B
- Authority
- CN
- China
- Prior art keywords
- ala
- pro
- lys
- boc
- arg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 75
- 238000002360 preparation method Methods 0.000 title abstract description 65
- 102000015636 Oligopeptides Human genes 0.000 title abstract description 14
- 108010038807 Oligopeptides Proteins 0.000 title abstract description 14
- 230000002537 thrombolytic effect Effects 0.000 title abstract description 7
- 239000003146 anticoagulant agent Substances 0.000 title abstract 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 25
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 18
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 11
- FYQSMXKJYTZYRP-DCAQKATOSA-N Pro-Ala-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 FYQSMXKJYTZYRP-DCAQKATOSA-N 0.000 claims description 10
- 238000009833 condensation Methods 0.000 claims description 10
- 125000006239 protecting group Chemical group 0.000 claims description 8
- MVEAAGBEUOMFRX-UHFFFAOYSA-N ethyl acetate;hydrochloride Chemical compound Cl.CCOC(C)=O MVEAAGBEUOMFRX-UHFFFAOYSA-N 0.000 claims description 7
- 230000005494 condensation Effects 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 claims description 6
- 239000003527 fibrinolytic agent Substances 0.000 claims description 3
- 229960000103 thrombolytic agent Drugs 0.000 claims description 3
- DICWIJISMKZDDY-VIFPVBQESA-N 1-o-tert-butyl 2-o-(2,5-dioxopyrrolidin-1-yl) (2s)-pyrrolidine-1,2-dicarboxylate Chemical compound CC(C)(C)OC(=O)N1CCC[C@H]1C(=O)ON1C(=O)CCC1=O DICWIJISMKZDDY-VIFPVBQESA-N 0.000 claims description 2
- 238000007127 saponification reaction Methods 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims 2
- 239000000825 pharmaceutical preparation Substances 0.000 claims 2
- 239000003937 drug carrier Substances 0.000 claims 1
- 238000009472 formulation Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 3
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 33
- 208000007536 Thrombosis Diseases 0.000 description 31
- 239000007787 solid Substances 0.000 description 28
- 238000005481 NMR spectroscopy Methods 0.000 description 20
- 239000000243 solution Substances 0.000 description 18
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 15
- 239000007864 aqueous solution Substances 0.000 description 14
- 230000000694 effects Effects 0.000 description 14
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 9
- 239000004698 Polyethylene Substances 0.000 description 9
- -1 polyethylene Polymers 0.000 description 9
- 229920000573 polyethylene Polymers 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 6
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 6
- 239000012230 colorless oil Substances 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- IWZSHWBGHQBIML-ZGGLMWTQSA-N (3S,8S,10R,13S,14S,17S)-17-isoquinolin-7-yl-N,N,10,13-tetramethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-amine Chemical compound CN(C)[C@H]1CC[C@]2(C)C3CC[C@@]4(C)[C@@H](CC[C@@H]4c4ccc5ccncc5c4)[C@@H]3CC=C2C1 IWZSHWBGHQBIML-ZGGLMWTQSA-N 0.000 description 5
- 230000006837 decompression Effects 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 238000001990 intravenous administration Methods 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- FYQSMXKJYTZYRP-UHFFFAOYSA-N 6-amino-2-[2-(pyrrolidine-2-carbonylamino)propanoylamino]hexanoic acid Chemical compound NCCCCC(C(O)=O)NC(=O)C(C)NC(=O)C1CCCN1 FYQSMXKJYTZYRP-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 4
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 229960005356 urokinase Drugs 0.000 description 4
- 210000001367 artery Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 210000001168 carotid artery common Anatomy 0.000 description 3
- 229920001971 elastomer Polymers 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 210000004731 jugular vein Anatomy 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- SITWEMZOJNKJCH-WDSKDSINSA-N Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N SITWEMZOJNKJCH-WDSKDSINSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- OPINTGHFESTVAX-BQBZGAKWSA-N Gln-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N OPINTGHFESTVAX-BQBZGAKWSA-N 0.000 description 2
- JLXVRFDTDUGQEE-YFKPBYRVSA-N Gly-Arg Chemical compound NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N JLXVRFDTDUGQEE-YFKPBYRVSA-N 0.000 description 2
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- KBPLFHHGFOOTCA-UHFFFAOYSA-N caprylic alcohol Natural products CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 125000004772 dichloromethyl group Chemical group [H]C(Cl)(Cl)* 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 2
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical group C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000001467 acupuncture Methods 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 210000001715 carotid artery Anatomy 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000002356 laser light scattering Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 208000020442 loss of weight Diseases 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N n-Octanol Natural products CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 210000004761 scalp Anatomy 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 238000002444 silanisation Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及用于溶血栓的寡肽化合物及其制备方法和应用。本发明的溶血栓寡肽化合物如以下结构所示。本发明通过动物实验评价了本发明寡肽化合物的溶血栓活性,证明了本发明的寡肽化合物除具有优秀的溶血栓活性外,还具有自主装性能。AA-Arg-Pro-Ala-Lys-R-CH2(CH2)12CH3
Description
技术领域
本发明涉及具有溶血栓功能的寡肽化合物及其制备方法和应用,属于生物医药领域。
背景技术
P6A(ARPAK)为纤维蛋白β链降解产物之一,具有溶血栓活性。在P6A的代谢研究中发现了代谢产物RPAK和PAK。在大鼠动静脉旁路插管溶血栓模型上,RPAK和PAK也具有溶血栓活性,且PAK的溶血栓活性比母体P6A强。按照一般的认识,多肽在体内都会迅速降解。通过PAK肽的结构修饰延缓体内降解速率和提高溶血栓活性,是寡肽溶血栓药物研究的重要途径。
按照一般的认识,含多肽的两亲性分子,例如脂肪醇链修饰的多肽,在适当的条件下通过分子间非共价键相互作用可发生自组装,形成纳米结构。借助纳米结构可改善多肽在体内的输送、延缓多肽在体内的降解速率和提高多肽的体内活性。
发明内容
本发明的目的在于提供具有溶血栓活性的寡肽化合物,并通过动物实验评价这些寡肽化合物的溶血栓活性,证明了本发明的溶血栓寡肽化合物除具有优秀的溶血栓活性外,还具有自主装性能。
本发明是通过以下技术方案来实现本发明的:
本发明的目的之一是提供Pro-Ala-Lys-R-CH2(CH2)12CH3,其中R为NH或O;提供Arg-Pro-Ala-Lys-R-CH2(CH2)12CH3,其中R为NH或O以及提供AA-Arg-Pro-Ala-Lys-R-CH2(CH2)12CH3,其中AA为Ala、Gly或Gln,R为NH或O。
本发明还提供了制备以上所述结构化合物的方法,具体包括如下步骤:
(1)在二环己基碳二亚胺(DCC)存在下Boc-Pro在无水THF中与N-羟基琥珀酰亚胺(HOSu)缩合为Boc-Pro-Osu,在NaHCO3存在下Boc-Pro-OSu与Ala反应生成Boc-Pro-Ala;
(2)在DCC和HOBt存在下Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl;
(3)在NaOH存在下,在甲醇中将Boc-Pro-Ala-Lys(Z)-OBzl皂化为Boc-Pro-Ala-Lys(Z);
(4)在DCC和HOBt存在下Boc-Pro-Ala-Lys(Z)在无水THF中与R1CH2(CH2)12CH3缩合为Boc-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3,其中R1为NH2或OH,R2为NH或O;
(5)在氯化氢-乙酸乙酯溶液中Boc-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3脱去保护基生成Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3;
(6)在乙醇、Pd/C和H2存在下Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3脱去保护基得到Pro-Ala-Lys-R2CH2(CH2)12CH3,其中R2为NH或O。
进一步,将上述步骤(5)获得的Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3进行以下三步反应,可获得Arg-Pro-Ala-Lys-RCH2(CH2)12CH3,其中R为NH或O,具体为:
(1)在DCC和HOBt存在下Boc-Arg(NO2)在无水THF中与Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3缩合为Boc-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3,其中,R2为NH或O;(由于该步骤的结构式中用的是R2,因此,采用了原来的修改,烦请确认)
(2)在氯化氢-乙酸乙酯溶液中Boc-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2-(CH2)12CH3脱去Boc生成Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3;
(3)在乙醇、Pd/C和H2存在下Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3脱去保护基,得到Arg-Pro-Ala-Lys-R2CH2(CH2)12CH3,其中R2为NH或O。
更进一步,将以上获得的Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3再进行以下三步反应可获得AA-Arg-Pro-Ala-Lys-RCH2(CH2)12-CH3,其中,AA为Ala、Gly或Gln残基,R为NH或O,具体为:
(1)在DCC和HOBt存在下Boc-AA在无水THF中与Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3缩合为Boc-AA-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2-(CH2)12CH3,其中AA为Ala、Gly或Gln残基;
(2)在氯化氢-乙酸乙酯溶液中Boc-AA-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3脱去Boc生成AA-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3;
(3)在乙醇、Pd/C和H2存在下AA-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2-(CH2)12CH3脱去保护基得到AA-Arg-Pro-Ala-Lys-R2CH2(CH2)12CH3,其中AA为Ala、Gly或Gln,R2为NH或O。
本发明通过动物实验评价了本发明寡肽化合物的溶血栓活性,证明了本发明寡肽化合物除具有优秀的溶血栓活性外,还具有自主装性能。
附图说明
图1为本发明寡肽化合物的合成路线图;
图2为本发明化合物19在水溶液中形成的纳米球的透射电镜照片。
在图1中,i)DCC、HOSu、NaHCO3和Ala;ii)DCC、HOBt、NMM;iii)氯化氢/乙酸乙酯溶液(4N);iv)NaOH水溶液;v)乙醇、Pd/C(5%)、H2(0.02Mba)。
具体实施方式
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
实施例1Boc-Pro-Ala的制备
将1.075g(5.0mmol)Boc-Pro溶于20mL无水THF,冰浴下向溶液中加入0.637g(5mmol)N-羟基琥珀酰亚胺(HOSu),并使完全溶解。冰浴下,把已溶于少量无水THF的二环己基碳二亚胺(DCC)1.236g(6.0mmol)加入反应液中。室温搅拌7h,TLC(氯仿/甲醇,10∶1)监测Boc-Pro消失。滤除二环己基脲(DCU),滤液减压浓缩除去THF。浓缩物用乙酸乙酯(EA)溶解,依次用饱和NaHCO3水溶液洗、饱和NaCl水溶液洗,然后将EA层减压浓缩至干,加入适量THF溶解。再加入已溶于少量水的Ala 0.489g(5.5mmol),用NaHCO3固体调pH到8-9,常温反应12h,减压浓缩除去THF,加入5mL水溶解,用饱和KHSO4水溶液调pH到2,用EA少量多次萃取,合并EA层,用饱和NaCl水溶液洗至中性,无水硫酸钠干燥。过滤,滤液减压浓缩至干,得1.41g(96%)标题化合物,为白色固体。ESI-MS(m/e):285[M-H]-;
实施例2Boc-Pro-Ala-Lys(Z)-OBzl的制备
将1.43g(5.0mmol)Boc-Pro-Ala溶于20mL无水THF,冰浴下往得到的溶液中加入0.675g(5mmol)N-羟基苯并三氮唑(HOBt),并使完全溶解。10分钟后加入1.071g(6mmol)二环己基碳二亚胺(DCC),得到反应液(I),待用。冰浴下把2.71g(5.0mmol)Lys(Z)-OBzl悬浮于20mL无水DMF中,然后加入1mL N-甲基吗啉(NMM),调pH 8-9。搅拌35分钟,得到反应液(II),待用。冰浴下把反应液(II)加入反应液(I)中,先冰浴下搅拌1h,再室温搅拌12h,TLC(氯仿/甲醇,10∶1)显示Lys(Z)-OBzl消失。滤除二环己基脲(DCU),滤液除去DMF。残留物用50mL氯仿溶解。得到的溶液依次用饱和NaHCO3水溶液洗、饱和NaCl水溶液洗、5%KHSO4水溶液洗和饱和NaCl水溶液洗。氯仿层用无水Na2SO4干燥、过滤、滤液减压浓缩至干,得到3.09g(97%)标题化合物,为米黄色固体。ESI-MS(m/e):639[M+H]+.IR(KBr):3311,2936,2872,1679,1534,1455,1379,1248,1166.1H NMR(300MHz,CDCl3):δ/ppm=8.77(m,1H),8.12(m,1H),7.88(m,1H),7.37(m,10H),5.22(m,2H),5.15(m,2H),4.60(q,J=4.5Hz,1H),4.27(t,J=6.5Hz,1H),3.49(m,1H),3.43(m,1H),3.13(m,2H),1.90(m,2H),1.85(m,2H),1.82(m,2H),1.70(m,1H),1.50(m,2H),1.48(s,9H),1.35(d,J=6.5Hz,3H).
实施例3Boc-Pro-Ala-Lys(Z)的制备
将1.59g(3.0mmol)Boc-Pro-Ala-Lys(Z)-OBzl溶于15mL甲醇。冰浴下将得到的溶液用NaOH(2N)水溶液调pHl2并搅拌2h,TLC(二氯/甲醇,15∶1)监测Boc-Pro-Ala-Lys(Z)-OBzl消失。反应混合物用稀盐酸(2N)调pH 7,减压浓缩除甲醇。残留物用稀盐酸(2N)调pH 2,用乙酸乙酯萃取3次。合并乙酸乙酯层,用饱和NaCl水溶液洗至中性,无水Na2SO4干燥。过滤,滤液减压浓缩至干,得1.40g(85%)标题化合物,为无色固体。ESI-MS(m/e):547[M-H]-。
实施例4Boc-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3(1)的制备
按照实施例2的制备方法由2.74g(5mmol)Boc-Pro-Ala-Lys(Z)和0.925g(5mmol)CH3(CH2)12CH2NH2得3.20g(90%)标题化合物,为米黄色固体。ESI-MS(m/e):743[M+H]+.Mp:105.3-105.7℃.IR(KBr):3277,2926,2852,1689,1635,1541,1452,1393,1367,1257,1163,1118,736,721,698.1H NMR(300MHz,CDCl3):δ/ppm=7.35(m,5H),6.33(d,J=6.6Hz,1H),5.2(t,J=12.0Hz,2H),4.3(t,J=4.2Hz,2H),4.19(t,J=6.0Hz,1H),3.4(m,2H),3.2(m,4H),1.7(m,10H),1-2(m,36H),0.89(t,J=6.9Hz,3H).
实施例5Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3的制备
将0.743g(1.0mmol)Boc-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3溶解在10mL4M氯化氢-乙酸乙酯溶液中,冰浴搅拌2小时,TLC(二氯/甲醇,15/1)监测原料点消失,减压浓缩除去乙酸乙酯,残留物反复加少量乙醚进行减压浓缩以除去氯化氢气体,最后得0.568g(91%)标题化合物,为米黄色固体。ESI-MS(m/e):643[M+H]+.
实施例6Pro-Ala-Lys-NHCH2(CH2)12CH3(3)的制备
将0.633g(1mmol)Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3置于50ml茄形瓶中,用甲醇溶解,加49mg Pd/C(5%),通H2(0.02Mba),室温搅拌至原料点消失。滤除Pd/C、滤液减压浓缩至干得0.38g(80%)标题化合物,为无色固体。ESI-MS(m/e):509[M+H]+.Mp:118.4-119.0℃.IR(KBr):3053,2951,2852,1739,1653,1531,1388,1242,1201,719.1H NMR(300MHz,CDCl3):δ/ppm=8.15(d,J=8.0Hz,3H),4.29(m,2H),3.10(m,3H),2.73(m,4H),2.30(s,3H),1.78(m,6H),1.55(m,4H),1.37(t,J=6.0Hz,3H),1.25(m,24H),0.86(t,J=7.0Hz,3H).
实施例7Boc-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3(2)的制备
按照实施例2的制备方法由2.74g(5mmol)Boc-Pro-Ala-Lys(Z)和0.93g(5mmol)CH3(CH2)12CH2OH得3.24g(87%))标题化合物,为米黄色固体。ESI-MS(m/e):744[M+H]+.Mp:90.3-91.8℃.IR(KBr):3294,2929,2856,1697,1531,1454,1390,1365,1249,1165,1026,777,738,698.1HNMR(300MHz,CDCl3):δ/ppm=7.18(m,5H),6.56(t,J=7.2Hz,1H),5.2(m,3H),4.5(m,5H),3.42(m,4H),1.5-2(m,10H),1.48(m,36H),0.89(t,J=6.9Hz,3H).
实施例8Pro-Ala-Lys(Z)-OCH2(CH2)12CH3的制备
按照实施例5的制备方法,从0.716g(1.0mmol)Boc-Pro-Ala-Lys(Z)-OCH2-(CH2)12CH3制得0.573g(93%)标题化合物,为米黄色固体。ESI-MS(m/e):644[M+H]+.
实施例9Pro-Ala-Lys-OCH2(CH2)12CH3(4)的制备
按照实施例6的制备方法由0.616g(1mmol)Pro-Ala-Lys(Z)-OCH2-(CH2)12CH3制得0.49g(80%)标题化合物,为无色固体。ESI-MS(m/e):510[M+H]+.Mp:121.9-122.5℃.IR(KBr):3255,3055,2924,2854,1735,1654,1541,1454,1384,1164,725.1H NMR(300MHz,CDCl3):δ/ppm=8.46(m,2H),4.20(m,2H),4.03(m,2H),3.36(s,1H),2.72(t,J=6.0Hz,4H),2.01(s,3H),1.91(m,4H),1.66(t,J=5.5Hz,6H),1.38(t,J=6.5Hz,3H),1.28(m,24H),0.86(t,J=7.0Hz,3H).
实施例10Boc-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3(5)的制备
按照实施例2的制备方法由3.08g(5mmol)Pro-Ala-Lys(Z)-NHCH2(CH2)12-CH3和1.6g(5.0mmol)Boc-Arg(NO2)制得3.67g(80%)标题化合物,为无色固体。ESI-MS(m/e):944[M+H]+.Mp:98.0-98.9℃.IR(KBr):3280,2926,2852,1708,1635,1529,1452,1390,1365,1253,1166,1052,1001,777,734,698.1H NMR(300MHz,CDCl3):δ/ppm=7.39(m,5H),6.63(s,1H),4-5(m,4H),3.6(m,2H),3.2(m,4H),2.1(m,5H),1-2(m,44H),0.89(t,J=6.9Hz,3H).
实施例11Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3的制备
按照实施例5的制备方法,从0.917g(1.0mmol)Boc-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3制得0.776g(95%)标题化合物,为无色固体。ESI-MS(m/e):844[M+H]+.
实施例12Arg-Pro-Ala-Lys-NHCH2(CH2)12CH3(7)的制备
按照实施例6的制备方法由0.817g(1mmol)Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3制得0.445g(70%)标题化合物,为无色固体。ESI-MS(m/e):665[M+H]+.Mp:102.2-104.7℃.IR(KBr):2926,2850,2794,1639,1533,1452,1369,1242,1159,1049,723.1H NMR(300MHz,CDCl3):δ/ppm=8.28(d,J=4.5Hz,5H),7.91(d,J=9.0Hz,1H),6.7(m,2H),4.43(s,3H),3-4(m,5H),2.72(d,J=11.5Hz,6H),2.01(s,5H),1.5-2.0(m,14H),1.38(s,3H),1.28(d,J=7.0Hz,20H),0.86(t,J=7.0Hz,3H).
实施例13Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3(6)的制备
按照实施例2的制备方法由3.08g(5mmol)Pro-Ala-Lys(Z)-OCH2(CH2)12CH3和1.6g(5.0mmol)Boc-Arg(NO2)制得3.90g(85%)标题化合物,为无色固体。ESI-MS(m/e):945[M+H]+.Mp:89.8-90.1℃.IR(KBr):3280,2926,2852,1708,1635,1529,1452,1390,1365,1253,1166,1052,1001,777,734,698.1H NMR(300MHz,CDCl3):δ/ppm=7.47(m,5H),6-7(m,1H),4-5(m,4H),3.6(m,2H),3.2(m,4H),2.1(m,5H),1-2(m,46H),0.89(t,J=6.0Hz,3H).
实施例14Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3的制备
按照实施例5的制备方法,从0.917g(1.0mmol)Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3制得0.776g(95%)标题化合物,为无色固体。ESI-MS(m/e):845[M+H]+.
实施例15Arg-Pro-Ala-Lys-OCH2(CH2)12CH3(8)的制备
按照实施例6的制备方法由0.818g(1mmol)Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3制得0.60g(84%)标题化合物,为无色固体。ESI-MS(m/e):666[M+H]+.Mp:122.8-123.2℃.IR(KBr):2924,2852,1743,1654,1525,1456,1240,1161,1043,721.1H NMR(300MHz,CDCl3):δ/ppm=8.3-9(m,2H),8.37(d,J=3.0Hz,2H),7.47(s,1H),6.63(s,2H),4.17(d,J=5.5Hz,5H),3.51(m,3H),2.73(t,J=5.5Hz,4H),2.35(d,J=6.0Hz,2H),2(s,5H),1.91(s,2H),1.58(m,8H),1.36(d,J=14.0Hz,27H),0.86(t,J=7.0Hz,3H).
实施例16Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3(9)的制备
按照实施例2的制备方法由0.945g(5mmol)Boc-Ala和4.09g(5.0mmol)Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3制得3.77g(77%)标题化合物,为无色固体。ESI-MS(m/e):1016[M+H]+.Mp:133.5-134.1℃.IR(KBr):3315,2929,2856,1632,1533,1454,1395,1367,1253,1165,1066,740,721,696.1H NMR(300MHz,CDCl3):δ/ppm=8.22(m,4H),7.36(m,5H),4-5(m,9H),3.40(m,4H),2.45(m,7H),1-2(m,49H),0.88(t,J=6.0Hz,3H).
实施例17Ala-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3的制备
按照实施例5的制备方法,从1.016g(1.0mmol)Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3制得0.719g(81%)标题化合物,为无色固体。ESI-MS(m/e):916[M+H]+.
实施例18Ala-Arg-Pro-Ala-Lys-NHCH2(CH2)12CH3(15)的制备
按照实施例6的制备方法由0.953g(1mmol)Ala-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3制得0.59g(73%)标题化合物,为无色油状物。ESI-MS(m/e):736[M+H]+.Mp:102.2-104.7℃.IR(KBr):3055,2929,2856,1737,1624,1533,1375,1238,1168,1035,725.1H NMR(300MHz,CDCl3):δ/ppm=8.45(d,J=7.2Hz,2H),8.21(m,3H),6.63(s,1H),4.31(m,4H),4.13(s,2H),3-4(m,3H),2.69(m,6H),2.0(d,J=1.8Hz,5H),1.81(m,6H),1.55(d,J=6.6Hz,7H),1.31(m,25H),0.85(t,J=7.2Hz,3H).
实施例19Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3(10)的制备
按照实施例2的制备方法由0.875g(5mmol)Boc-Gly和4.09g(5.0mmol)Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3制得3.41g(70%)标题化合物,为无色固体。ESI-MS(m/e):1002[M+H]+.Mp:94.0-94.6℃.IR(KBr):3329,2929,2856,1654,1529,1452,1392,1367,1247,1163,1051,1026,748,721,698.1H NMR(300MHz,CDCl3):δ/ppm=8.22(m,4H),7.36(m,5H),4-5(m,9H),3.40(m,4H),2.45(m,7H),1-2(m,49H),0.88(t,J=6.0Hz,3H).
实施例20Gly-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3的制备
按照实施例5的制备方法,从0.974g(1.0mmol)Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3制得0.725g(81%)标题化合物,为无色固体。ESI-MS(m/e):902[M+H]+.
实施例21Gly-Arg-Pro-Ala-Lys-NHCH2(CH2)12CH3(16)的制备
按照实施例6的制备方法由0.874g(1mmol)Gly-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3制得0.52g(73%)标题化合物,为无色油状物。ESI-MS(m/e):722[M+H]+.Mp:144-145℃.1H NMR(300MHz,CDCl3):δ/ppm=8.33(d,J=6.9Hz,4H),7.84(t,J=4.8Hz,1H),5-7(m,4H),4.31(t,J=4.2Hz,4H),3.59(m,7H),2.72(d,J=4.8Hz,6H),2.05(s,5H),1.81(m,4H),1.55(s,6H),1.36(m,27H),0.88(t,J=6.0Hz,3H).
实施例22Boc-Gln-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3(11)的制备
按照实施例2的制备方法由1.23g(5mmol)Boc-Gln和4.09g(5.0mmol)Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3制得4.33g(82%)标题化合物,为无色固体。ESI-MS(m/e):1085[M+H]+.Mp:90.9-91.6℃.IR(KBr):3325,2926,2850,1697,1627,1573,1535,1448,1392,1367,1311,1159,1087,732,696.1H NMR(300MHz,CDCl3):δ/ppm=8.72(m,4H),7.26(m,5H),4-5(m,9H),3.40(m,4H),2.45(m,7H),1-2(m,49H),0.88(t,J=6.0Hz,3H).
实施例23Gln-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3的制备
按照实施例5的制备方法,从1.045g(1.0mmol)Boc-Gln-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3制得0.822g(87%)标题化合物,为无色固体。ESI-MS(m/e):985[M+H]+.
实施例24Gln-Arg-Pro-Ala-Lys-NHCH2(CH2)12CH3(17)的制备
按照实施例6的制备方法由0.985g(1mmol)Gln-Arg(NO2)-Pro-Ala-Lys(Z)-NHCH2(CH2)12CH3制得0.499g(63%)标题化合物,为无色油状物。ESI-MS(m/e):793[M+H]+.Mp:131-132℃.IR(KBr):2926,2848,1625,1573,1448,1311,1242,1069,893,721,640.1H NMR(300MHz,CDCl3):δ/ppm=7.9(m,9H),6.43(s,2H),4-5(m,4H),3-4(m,5H),2.51(m,6H),2.01(t,J=5.0Hz,9H),1-2(m,37H),0.86(t,J=5.0Hz,3H).
实施例25Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3(12)的制备
按照实施例2的制备方法由0.945g(5mmol)Boc-Ala和4.09g(5.0mmol)Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3制得3.89g(74%)标题化合物,为无色固体。ESI-MS(m/e):1016[M+H]+.Mp:78-79℃.IR(KBr):3313,2933,2868,1631,1537,1452,1392,1367,1251,1166,1051,1026,750,738,696.1H NMR(300MHz,CDCl3):δ/ppm=8.39(m,3H),7.30(m,5H),6.68(m,3H),5.12(m,2H),4.53(m,5H),4.09(t,J=6.5Hz,2H),3.62(m,2H),3.13(m,2H),2.25(s,7H),1.71(m,6H),1.53(m,12H),1.30(m,31H),0.90(t,J=7.0Hz,3H).
实施例26Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3的制备
按照实施例5的制备方法,从0.989g(1.0mmol)Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3制得0.755g(81%)标题化合物,为无色固体。ESI-MS(m/e):916[M+H]+.
实施例27Ala-Arg-Pro-Ala-Lys-OCH2(CH2)12CH3(18)的制备
按照实施例6的制备方法由0.917g(1mmol)Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3制得0.68g(74%)标题化合物,为无色油状物。ESI-MS(m/e):738[M+H]+.Mp:80.6-81.7℃.IR(KBr):3055,2951,2852,1739,1653,1550,1456,1375,1251,1161,719.1H NMR(300MHz,CDCl3):δ/ppm=8.9(m,5H),7.95(s,1H),6.6(s,2H),4.35(d,J=5.1Hz,6H),3.64(m,3H),2.73(s,6H),2.0(s,5H),1.78(m,4H),1.59(s,6H),1.31(m,30H),0.85(s,3H).
实施例28Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3(13)的制备
按照实施例2的制备方法由0.875g(5mmol)Boc-Gly和4.09g(5.0mmol)Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3制得3.41g(70%)标题化合物,为无色固体。ESI-MS(m/e):1002[M+H]+.Mp:88-89℃.IR(KBr):3313,2933,2868,1631,1537,1452,1392,1367,1251,1166,1051,1026,750,738,696.1H NMR(300MHz,CDCl3):δ/ppm=8.45(m,3H),7.30(m,5H),6.68(m,3H),5.12(m,2H),4.53(m,5H),4.09(t,J=6.5Hz,2H),3.62(m,2H),3.13(m,2H),2.25(s,7H),1.71(m,6H),1.53(m,12H),1.30(m,31H),0.90(t,J=7.0Hz,3H).
实施例29Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3的制备
按照实施例5的制备方法从1.002g(1.0mmol)Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3制得0.787g(86%)标题化合物,为无色固体。ESI-MS(m/e):900[M+H]+.
实施例30Gly-Arg-Pro-Ala-Lys-OCH2(CH2)12CH3(19)的制备
按照实施例6的制备方法由0.875g(1mmol)Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3制得0.520g(75%)标题化合物,为无色油状物,易吸潮。ESI-MS(m/e):723[M+H]+.Mp:75-76℃.IR(KBr):2924,2858,1737,1624,1544,1448,1404,1242,1207,1049,719.1H NMR(300MHz,CDCl3):δ/ppm=7.9(m,4H),6.63(s,2H),5.12(m,2H),4-5(m,6H),3-4(m,5H),2.37(m,6H),2.01(s,5H),1.82(m,10H),0.86(t,J=5.0Hz,3H).
实施例31Boc-Gln-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3(14)的制备
按照实施例2的制备方法由1.23g(5mmol)Boc-Gln和4.09g(5.0mmol)Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3制得4.18g(80%)标题化合物,为无色固体。ESI-MS(m/e):1073[M+H]+.Mp:98-99℃.IR(KBr):3313,2933,2868,1631,1537,1452,1392,1367,1251,1166,1051,1026,750,738,696.1H NMR(300MHz,CDCl3):δ/ppm=8.44(m,3H),7.38(m,5H),6.87(m,3H),5.12(m,2H),4.53(m,5H),4.09(t,J=6.5Hz,2H),3.62(m,2H),3.13(m,2H),2.25(s,7H),1.71(m,6H),1.53(m,12H),1.30(m,31H),0.90(t,J=7.0Hz,3H).
实施例32Gln-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3的制备
按照实施例5的制备方法,从1.046g(1.0mmol)Boc-Gln-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3制得0.804g(83%)标题化合物,为无色固体。ESI-MS(m/e):973[M+H]+.
实施例33Gln-Arg-Pro-Ala-Lys-OCH2(CH2)12CH3(20)的制备
按照实施例6的制备方法由0.973g(1mmol)Gln-Arg(NO2)-Pro-Ala-Lys(Z)-OCH2(CH2)12CH3制得0.517g(64%)标题化合物,为无色油状物。ESI-MS(m/e):793[M+H]+.Mp:136-137℃.IR(KBr):2920,2818,1735,1618,1543,1448,1408,1110,997,721.1H NMR(300MHz,CDCl3):δ/ppm=8.47(s,5H),7.37(m,3H),6.68(s,2H),4-5(m,6H),3.37(m,3H),2.73(d,J=5.0Hz,6H),2.00(m,7H),1.7(m,10H),1.36(d,J=5.0Hz,23H),0.86(t,J=5.0Hz,3H).
实验例1化合物3,4,7,8,15-20静脉注射给药的体内溶血栓活性试验
1)评价方法
将200-220g雄性SD大鼠用20%乌拉坦溶液(6mL/kg,i.p.)进行麻醉。麻醉大鼠仰卧位固定,分离右颈总动脉,于近心端夹动脉夹,近心端和远心端分别穿入手术线,将远心端的手术线于皮毛用止血钳夹紧,在远心端插管,松开动脉夹,放出约1mL动脉血并装在1mL的EP管中。往垂直固定的玻璃管(长15mm,内径2.5mm,外径5.0mm,管底用胶塞密封)中注入0.1ml大鼠动脉血液,往管内迅速插入一支不锈钢质料的血栓固定螺栓。该血栓固定螺旋用直径为0.2mm的不锈钢丝绕成,螺旋部分长12mm,含15个螺圈,螺圈的直径为1.0mm,托柄与螺旋相连,长7.0mm,呈问号型。血液凝固15min后,打开玻璃管底部的胶塞,用镊子固定血栓固定螺旋的托柄,从玻璃管中取出被血栓包裹的血栓固定螺旋,精确称重。
旁路插管由3段构成,中段为聚乙烯胶管,长60mm,内径3.5mm,两端为相同的聚乙烯管,长100mm,内径1mm,外径2mm,该管的一端拉成尖管(用于插入大鼠颈动脉或静脉),外径1mm,另一端的外部套一段长7mm,外径3.5mm的聚乙烯管(加粗,用于插入中段的聚乙烯胶管内)。3段管的内壁均硅烷化。将血栓包裹的血栓固定螺旋放入中段聚乙烯胶管内,胶管的两端分别与两根聚乙烯的加粗端相套。用注射器通过尖管端将管中注满肝素生理盐水溶液(50IU/kg)备用。
分离大鼠的左颈外静脉,近心端和远心端分别穿入手术线,结扎远心端,在暴露的左颈外静脉上小心地剪一斜口,将上面制备好的旁路管道的尖管由斜口插入左颈外静脉开口的近心端,同时远离旁路管中段(含精确称重的血栓固定螺旋)内血栓固定螺旋的托柄。用注射器通过另一端的尖管推入准确量的肝素生理盐水(50IU/kg),此时注射器不撤离聚乙烯管,用止血钳夹住注射器与聚乙烯管之间的软管。在右颈总动脉的近心端用动脉夹止血,在离动脉夹不远处将右颈总动脉小心地剪一斜口。从聚乙烯管的尖部拔出注射器,将聚乙烯管的尖部插入动脉斜口的近心端。旁路管道的两端都用4号手术缝线与动静脉固定。
用头皮针将生理盐水,尿激酶的生理盐水溶液或不同浓度化合物的生理盐水溶液通过旁路管的中段(含精确称重的血栓固定螺旋),刺入远离血栓固定螺旋的近静脉处,打开动脉夹,使血流通过旁路管道从动脉流向静脉,此即大鼠动静脉旁路溶栓模型,缓慢将注射器中的液体注入到血液中(约6min),使生理盐水,尿激酶或本发明的化合物通过血液循环,按静脉-心脏-动脉的顺序作用到血栓上。从开始注射时计时,1h后从旁路管道中取出血栓固定螺旋,精确称重。计算每只大鼠旁路管道中血栓固定螺旋给药前后的质量差,统计并评价化合物的体内溶栓活性。血栓减重用均值和标准差表示。
2)给药方法和剂量
静脉注射给药;空白对照:生理盐水,给药剂量为3mL/kg;阳性对照:尿激酶,给药剂量为20000U/kg,相当于1.68mg/kg;本发明化合物的给药剂量为1.0nmol/kg.
3)评价结果如表1所示。
表1
表1为1.0nmol/kg化合物3,4,7,8,15-20经静脉注射给药的溶血栓活性a,其中,a)n=10,尿激酶剂量为20000U/kg;b)与生理盐水组比,P<0.01.
实验例2化合物19静脉注射给药的溶血栓量效关系
1)评价方法
同实验例1中1)的方法。
2)给药方法和剂量
静脉注射给药;选取高、中、低三个浓度考察化合物19的剂量效应依赖关系,剂量为1nmol/kg,0.1nmol/kg和0.01nmol/kg.
3)评价结果如表2所示。
表2
表2为静脉注射化合物19的量效关系a,其中,a)n=10;b)与0.1nmol/kg组比,p<0.01;c)与0.01nmol/kg组比,p<0.01;d)与生理盐水组比,P>0.05.
实验例3化合物3,4,7,8,15-20的自主装性能评价
在激光散射粒度仪上观察了25℃时水溶液和正辛醇中化合物3-4、7-8、15-20的粒径,浓度为1mg/mL,结果如表3所示。表3的数据表明,本发明化合物在水相和脂相中均具有自组装成为纳米球的性能。以化合物19为例,化合物19在水溶液中形成的纳米球的透射电镜照片如图2所示。
表3
Claims (10)
1.以下结构的化合物,
Pro-Ala-Lys-R-CH2(CH2)12CH3
其中R为NH或O。
2.以下结构的化合物,
Arg-Pro-Ala-Lys-R-CH2(CH2)12CH3
其中R为NH或O。
3.以下结构的化合物,
AA-Arg-Pro-Ala-Lys-R-CH2(CH2)12CH3
其中AA为Ala、Gly或Gln,R为NH或O。
4.一种制备权利要求1所述化合物的方法,其特征在于包括如下步骤:
(1)在二环己基碳二亚胺存在下Boc-Pro在无水THF中与N-羟基琥珀酰亚胺缩合为Boc-Pro-Osu,在NaHCO3存在下Boc-Pro-OSu与Ala反应生成Boc-Pro-Ala;
(2)在DCC和HOBt存在下Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl;
(3)在NaOH存在下,在甲醇中将Boc-Pro-Ala-Lys(Z)-OBzl皂化为Boc-Pro-Ala-Lys(Z);
(4)在DCC和HOBt存在下Boc-Pro-Ala-Lys(Z)在无水THF中与R1CH2(CH2)12CH3缩合为Boc-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3,其中R1为NH2或OH,R2为NH或O;
(5)在氯化氢-乙酸乙酯溶液中Boc-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3脱去保护基生成Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3;
(6)在乙醇、Pd/C和H2存在下Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3脱去保护基得到权利要求1所述化合物。
5.一种制备权利要求2所述化合物的方法,其特征在于包括如下步骤:
(1)在DCC和HOBt存在下Boc-Arg(NO2)在无水THF中与权利要求4的步骤(5)所得化合物缩合为Boc-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3,其中R2为NH或O;
(2)在氯化氢-乙酸乙酯溶液中Boc-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2-(CH2)12CH3脱去Boc生成Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3;
(3)在乙醇、Pd/C和H2存在下Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3脱去保护基,得到权利要求2所述化合物。
6.一种制备权利要求3所述化合物的方法,其特征在于,包括如下步骤:
(1)在DCC和HOBt存在下Boc-AA在无水THF中与权利要求5中步骤(2)所得化合物缩合为Boc-AA-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3;其中AA为Ala、Gly或Gln的残基,其中R2为NH或O;
(2)在氯化氢-乙酸乙酯溶液中Boc-AA-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3脱去Boc生成AA-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2(CH2)12CH3;
(3)在乙醇、Pd/C和H2存在下AA-Arg(NO2)-Pro-Ala-Lys(Z)-R2CH2-(CH2)12CH3脱去保护基,得到权利要求3所述的化合物。
7.一种药物组合物,其特征在于,含有治疗上有效剂量的权利要求1所述化合物或权利要求2所述化合物或权利要求3所述化合物,并含有一种或多种药学上可接受的赋型剂或者辅加剂。
8.一种药物制剂,其特征在于,将有效量的权利要求1所述化合物或权利要求2所述化合物或权利要求3所述化合物与药学上可接受的载体或稀释剂配合后,按本领域常规的制剂方法将其制备成任意一种适宜的药物制剂。
9.权利要求1-3中任一项所述化合物在制备溶血栓药物中的应用。
10.权利要求7所述的药物组合物在制备溶血栓药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201010576378.8A CN102485744B (zh) | 2010-12-01 | 2010-12-01 | 溶血栓的寡肽化合物及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201010576378.8A CN102485744B (zh) | 2010-12-01 | 2010-12-01 | 溶血栓的寡肽化合物及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN102485744A CN102485744A (zh) | 2012-06-06 |
CN102485744B true CN102485744B (zh) | 2014-10-29 |
Family
ID=46151319
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201010576378.8A Expired - Fee Related CN102485744B (zh) | 2010-12-01 | 2010-12-01 | 溶血栓的寡肽化合物及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN102485744B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101190941A (zh) * | 2006-11-30 | 2008-06-04 | 首都医科大学 | 具有溶血栓活性的多肽、其制备方法及应用 |
CN101367873A (zh) * | 2008-10-08 | 2009-02-18 | 南开大学 | 一种改构的胰高血糖素样肽-1的类似物和修饰物及其应用 |
CN101516400A (zh) * | 2006-08-11 | 2009-08-26 | 国立大学法人宫崎大学 | 以生长素释放肽及其衍生物或作用于GHS-R1a的物质作为有效成分的脊髓神经修复促进治疗剂 |
CN101538312A (zh) * | 2009-05-08 | 2009-09-23 | 首都医科大学 | Rgd-脂肪胺系列化合物作为肿瘤靶向载体材料的制备及应用 |
-
2010
- 2010-12-01 CN CN201010576378.8A patent/CN102485744B/zh not_active Expired - Fee Related
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101516400A (zh) * | 2006-08-11 | 2009-08-26 | 国立大学法人宫崎大学 | 以生长素释放肽及其衍生物或作用于GHS-R1a的物质作为有效成分的脊髓神经修复促进治疗剂 |
CN101190941A (zh) * | 2006-11-30 | 2008-06-04 | 首都医科大学 | 具有溶血栓活性的多肽、其制备方法及应用 |
CN101367873A (zh) * | 2008-10-08 | 2009-02-18 | 南开大学 | 一种改构的胰高血糖素样肽-1的类似物和修饰物及其应用 |
CN101538312A (zh) * | 2009-05-08 | 2009-09-23 | 首都医科大学 | Rgd-脂肪胺系列化合物作为肿瘤靶向载体材料的制备及应用 |
Also Published As
Publication number | Publication date |
---|---|
CN102485744A (zh) | 2012-06-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101190940B (zh) | 具有靶向抗血栓活性的多肽、其制备方法和应用 | |
CN103159835B (zh) | Lys及寡肽修饰的姜黄素衍生物、其合成及在医学中的应用 | |
RU2604193C2 (ru) | Новое соединение с эффектами тромболизиса, акцептирования свободных радикалов и направленного действия на тромб, а также способ его получения и применение | |
CN101190941B (zh) | 具有溶血栓活性的多肽、其制备方法及应用 | |
CN103998063B (zh) | 聚乙二醇基肾上腺髓质素前药及其用途 | |
CN102807605B (zh) | Nα-(1,3-二氧-4,4,5,5-四甲基咪唑啉-2-苯基-4’-氧乙酰基)-Nω-脂肪酰基-Lys-Arg-Gly-Asp-Phe、其制备方法和应用 | |
CN101190942B (zh) | 具有溶血栓活性的化合物、其制备方法和应用 | |
CN104610424B (zh) | Lys(Pro-Ala-Lys)姜黄素衍生物、其合成及在医学中的应用 | |
CN102477076B (zh) | 用于溶血栓的寡肽化合物及其制备方法和应用 | |
CN102485744B (zh) | 溶血栓的寡肽化合物及其制备方法和应用 | |
CN102807604B (zh) | Nα-(1,3-二氧-4,4,5,5-四甲基咪唑啉-2-苯基-4’-氧乙酰基)-Nω-脂肪酰基-Lys-Arg-Gly-Asp-Val、其制备方法和应用 | |
CN102485745B (zh) | 溶血栓寡肽及其制备方法和应用 | |
CN102485747B (zh) | 具有靶向性溶血栓活性的寡肽、其制备方法和应用 | |
CN102485746B (zh) | 具有靶向性溶血栓活性的寡肽、其制备方法和应用 | |
CN102477075B (zh) | 用于抗血栓的寡肽及其制备方法和应用 | |
CN1978462B (zh) | 咪唑啉修饰的氨基酸,其合成方法及在多肽标记中的应用 | |
CN102485748B (zh) | 具有靶向性溶血栓活性的寡肽、其制备方法和应用 | |
CN102796168B (zh) | 一种具有溶血栓活性的化合物,及其制备方法和应用 | |
CN108929372A (zh) | 1R-甲基-β-四氢咔啉酰-K(GRPAK)-RGDV,其合成,活性和应用 | |
CN108948146A (zh) | 1R-甲基-β-四氢咔啉酰-K(ARPAK)-RGDV, 其合成, 活性和应用 | |
CN102477069B (zh) | Pro-Ala-Lys-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3、及其合成和作为溶血栓剂的应用 | |
CN103159826B (zh) | Lys及Lys(Pro-Ala-Lys)修饰的姜黄素衍生物、其合成及在医学中的应用 | |
CN102477072B (zh) | 用于溶血栓的寡肽化合物及其制备方法和应用 | |
CN108948155A (zh) | 1R-甲基-β-四氢咔啉酰-K(QRPAK)-RGDV, 其合成, 活性和应用 | |
CN102477071B (zh) | Pro-Ala-Lys-Lys(Pro-Ala-Lys)-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3、其合成和作为溶血栓剂的应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141029 Termination date: 20181201 |